Influenza h5 vaccines

ABSTRACT

The present invention is based on the surprising finding that H5 protein of clade 1 H5N1 induces, in particular by a single-shot vaccination, a cross-clade protective immune response to influenza viruses with H5N1 HA. In one aspect, the invention is thus directed to H5 protein of clade 1 H5N1 virus for use in a method of treating or preventing infections with H5N1 virus of a different clade, namely of a clade different from clade 1 or from any clade with the exception of clade 1, respectively.

FIELD OF THE INVENTION

The present invention relates to the field of medicine, preferably tothe field of infectious diseases. In particular the present inventionrelates to influenza proteins and vaccines. Most particularly, thepresent invention relates to the use of any of such proteins or vaccinesfor the treatment and prevention of influenza infections, furthermorefor the prevention of intra- and inter-species transmission of influenzavirus.

BACKGROUND OF THE INVENTION

Influenza infection remains an important infection in animals andhumans. Influenza is caused by viruses that undergo continuous antigenicchanges/modifications and that possess an animal reservoir. Thus newepidemics and pandemics may occur in the future, and eradication of thedisease will be difficult to achieve. Influenza viruses are well knownin the art and described more in detail for example by P. Palese, NatureMedicine, vol. 10, no. 12, pp. S 82 to S 86 of December 2004, withfurther references. Briefly, the genome of the influenza A virusconsists of eight single-stranded segments, and the viral particles hastwo major glycoproteins on its surface: hemagglutinin (H) andneuraminidase (N). With at least 16 different hemagglutinin (H1 to H16)and 9 different neuraminidase (N1 to N9) subtypes, there is aconsiderable antigenic variation among influenza viruses.

Influenza virus of type H5N1 Fowl Plague virus has been demonstrated toinfect poultry, pigs and man. The viruses can also be transmitteddirectly from avian species to humans (Claas et al., Lancet 1998, 351:472; Suarez et al., J. Virol. 1998, 72: 6678; Subbarao et al., Science1998, 279: 393; Shortridge, Vaccine 1999, 17 (Suppl. 1): S26-S29).Mortality in known human clinical cases approaches about 50%.

Over the last century pigs have been an important vector for influenzapandemics. Pigs, camels, and seals, preferably pigs, can serve as a‘mixing chamber’ for avian influenza viruses, and therefore represent apotential risk factor for overcoming the species hurdles from poultry,the naturally reservoir of influenza viruses, to mammals. This normallyoccurs by double infections of the susceptible animals, e.g. pig, withboth, an established mammalian (porcine), as well as an avian influenzavirus. This double infection may create new recombinant viruses that maybe the cause of human or porcine pandemics. Recent evidence would,however, indicate that a recombination of current avian H5 strains withmammalian influenza viruses will not result in highly virulentrecombinants. On the other hand, avian influenza virus can infect pigsand by spontaneous mutations can become adapted to pigs. The criticalhurdle will be overcome as soon as the virus can cause horizontalinfections within a pig (or other mammalian) population.

Yet, a major part of Southeast Asian pigs have been infected with avian(H5) influenza virus strains originating from neighbouring poultryhusbandry. As those infections have so far been sub-clinical, they canonly be diagnosed by laboratory methods and thus are frequentlyoverlooked. There is a high risk that those sub-clinically-infected pigswill serve as an opportunity for the virus to adapt to the mammaliansystem, spread within the porcine population, and also infect humanbeings.

Current influenza vaccines include a subunit vaccine (Babai et al.,Vaccine 1999, 17(9-10):1223-1238; Crawford at al., Vaccine 1999,17(18):2265-2274; Johansson et al., Vaccine 1999, 17(15-16):2073-2080)attenuated vaccine (Horimoto et al., Vaccine 2004, 22(17-18):2244-2247),DNA vaccine (Watabe et al., Vaccine 2001, 19(31):4434-4444) andinactivated influenza vaccine (Cao at al., Vaccine 1992, 10(4):238-242),with the latter being the most widely used on a commercial scale(Lipatov at al., J Virol 2004, 78(17):8951-8959).

Subunit vaccines, recombinant hemagglutinin and neuraminidase (Babai etal., Vaccine 1999, 17(9-10):1223-1238; Crawford et al., Vaccine 1999,17(18):2265-2274; Johansson et al., Vaccine 1999, 17(15-16):2073-2080)may be an attractive alternative to the inactivated vaccine, althoughnone are currently in use as commercial vaccines. The preparation ofsuch vaccines is obviously safer than for an inactivated vaccine.Moreover, subunit vaccines do not generate antibody responses tointernal influenza viral proteins and thus allow distinction betweenvaccinated and infected animals (Crawford at al., Vaccine 1999,17(18):2265-2274).

Hemagglutinin protein is the receptor-binding and membrane fusionglycoprotein of influenza virus and the target forinfectivity-neutralizing antibodies. The entire hemagglutinin protein(HA) from the H5N1 is composed of 568 amino acids, with a molecularweight of 56 kDa. The HA molecule consists of HA1 and HA2 subunits, withthe HA1 subunit mediating initial contact with the cell membrane and HA2being responsible for membrane fusion (Chizmadzhev, Bioelectrochemistry2004, 63(1-2):129-136).

Baculovirus/insect cell systems have been used to express hemagglutiningenes isolated from avian influenza subtypes (Babai at al., Vaccine1999, 17(9-10):1223-1238; Crawford et al., Vaccine 1999,17(18):2265-2274; Johansson et al., Vaccine 1999, 17(15-16):2073-2080);Nwe et al., BMC Mircobiology 2006, 6(16):doi:10.1186/1471-2180-6-16).However, those recombinant proteins seem not to be protective in anycase, or only less effective at least for some species (Treanor et al.,Vaccine 2001, 19: 1732-1737).

The document Lin et al. (J Vet Med Sci. 2008 70(11):1147-52) disclosesthe use of a baculovirus/insect cell system for the production of H5protein of clade 2 H5N1 virus A/duck/China/E319-2/03, which is usablefor a prime-booster vaccination for preventing an infection with theclade 2 virus A/duck/China/E319-2/03.

Bright at al. (PLoS One. 2008 30; 3(1):e1501) describes the use of abaculovirus/insect cell system for generating virus-like particles(VLPs) which include neuraminidase, hemagglutinin and matrix 1 proteinfrom clade 2 H5N1 virus for inducing a cross-clade protective immuneresponse against a challenge with clade 1 H5N1 virus A/VN/1203/2004 inmice. However, the production of VLPs is not without problems, since inorder to generate a functional VLP that effectively mimic a real virus,multiple virus structural proteins are needed which must then becorrectly assembled into a particle that reproduces the confirmation ofthe outer shell (capsid) of the infectious virus. Further, study alsoreals that in vitro assembly of VLPs competes with aggregation (Ding atal. Biotechnology and Bioengineering 107 (3): 550-560).

Thus, there is a need to increase availability of improved vaccines andnew vaccination approaches to provide better approaches to controlinfluenza infections and to have a positive impact on disease load. Inparticular, there is a strong need for a simple, effective andeasy-to-handle system inducing, preferably by a single-shot vaccination,a cross-clade protective immune response to influenza viruses with H5N1HA.

DESCRIPTION OF THE INVENTION

Before the embodiments of the present invention it shall be noted thatas used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a preparation” includes aplurality of such preparations; reference to the “carrier” is areference to one or more carriers and equivalents thereof known to thoseskilled in the art, and so forth. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. All given ranges and values may vary by 1 to 5%unless indicated otherwise or known otherwise by the person skilled inthe art, therefore, the term “about” was omitted from the description.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods, devices, and materials are nowdescribed. All publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing the substances,excipients, carriers, and methodologies as reported in the publicationswhich might be used in connection with the invention. Nothing herein isto be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

The solution to the above technical problem is achieved by thedescription and the embodiments characterized in the claims.

Influenza Proteins and Nucleic Acid Molecules Coding for Those

The present invention is based on the surprising finding that H5 proteinof clade 1 H5N1 induces, in particular by a single-shot vaccination, across-clade protective immune response to influenza viruses with H5N1HA. As one feature, the H5 protein of clade 1 H5N1 virus, which is forreasons of clarity also termed “H5 protein (1)” herein, comprises orconsists of a polypeptide sequence having at least 98% sequence identitywith the polypeptide sequence set forth in SEQ ID NO:1.

A “single-shot vaccination” refers to an immunogenic composition that iseffective at reducing the incidence of or severity of infection after asingle dose thereof, without the need for a booster.

In one aspect, the invention is thus directed to H5 protein (1) of clade1 H5N1 virus for use in a method of treating or preventing infectionswith H5N1 virus of a different clade, namely of a clade different fromclade 1 or from any clade with the exception of clade 1, respectively,wherein said H5 protein (1) comprises or consists of a polypeptidesequence having at least 98% sequence identity with the polypeptidesequence of SEQ ID NO:1.

The term “clade” or “clades” as used herein relates to the clade(s) ofthe WHO Nomenclature System for the highly Pathogenic Avian InfluenzaVirus (H5N1), which is summarized at the WHO website URL:who.int/csr/disease/avian_influenza/guidelines/nomencature/en/(12 Aug.2011), which is incorporated herein by reference.

10 distinct initial clades of viruses (numbered 0-9) are defined(WHO/OIE/FAO H5N1 Evolution Working Group, 2008), which are called firstorder clades. Clades are strictly defined on the nucleotide level asmeeting the following three specific clade definition criteria developedby the WHO/OIE/FAO H5N1 Evolution Working Group:

-   -   sharing of a common (clade-defining) node;    -   monophyletic grouping with a bootstrap value of ≧60 at the        clade-defining node (after 1000 neighbor-joining bootstrap        replicates); and    -   average percentage pairwise nucleotide distances between and        within clades of >1.5% and <1.5%, respectively.

As the viruses within these 10 clades continue to evolve, newsublineages (potential H5N1 clades) periodically emerge. Once thesesublineages meet the same three specific clade definition criteria asthe initial 10 clades (numbered 0-9), they are designated as separateclades (WHO/OIE/FAO H5N1 Evolution Working Group Emerg. Inf. Dis. 14, 7(2008). These new clades are defined as second (or third, etc) orderclades and assigned a numerical ‘address’ which links them to theiroriginal clade using a hierarchical decimal numbering system. Forexample, within the antigenically distinct clade 2.3, third order cladesmeeting the clade definition are designated as clades 2.3.1 and 2.3.2and so on. This logical hierarchal numbering system is objectivelyrelated to HA phylogeny.

The criteria used for the clade designation according to the WHONomenclature System for H₅Nlare:

-   -   1 Maintain previously designated clade numbers where possible        (i.e., clade 2.2 remains 2.2 and clade 1 remains 1)    -   2 New clade designations based on phylogenetic tree topology        derived from all available sequences (the large tree)        -   H5N1 progenitors (closest to Gs/Guangdong/1/96)            re-designated as clade 0        -   Subsequent clades numbered starting from clade 3 (i.e.,            clades 3-9)        -   Clades designated by presence of a distinct common node            shared by at least 4 isolates (in a monophyletic group)        -   Additional branches designated as a single clade evolves            into more than one distinct lineage (i.e., clade 2.2 or            clade 2.3.1; based on sharing of a common node and            monophyletic grouping)    -   3 Average percentage pairwise distances between and within        clades (using Kimura 2-parameter)        -   Distinct clades should have >1.5% average distances between            other clades        -   Distinct clades should have <1.5% average distances within            the clade (may be slightly higher in clades with highly            evolved outliers; i.e., Ck/Shanxi/2/2006 in clade 7)    -   4 Bootstrap (based on 1,000 neighbor-joining bootstrap        replicates) ≧60% bootstrap value at clade-defining node    -   (taken from Table 1 of: WHO/OIE/FAO H5N1 Evolution Working Group        Emerg. Inf. Dis. 14, 7 (2008)).

The prototype strain for each clade is listed in the following Table:

Clade Prototype strain 0 Gs/Guangdong/1/96 3 Ck/Hong Kong/YU562/2001 4Gs/Guiyang/337/2006 5 Gs/Guangxi/914/2004 6 Ck/Hunan/01/2004 7Ck/Shanxi/2/2006 8 Ck/Hong Kong/YU777/2002 9 Dk/Guangxi/2775/2005 1Vietnam/1203/2004 2.1.1 Ck/Indonesia/BL/2003 2.1.2 Indonesia/538H/20062.1.3 Indonesia/5/2005 2.2 BHGs/Qinghai/1A/2005 2.3.1 Dk/Hunan/303/20042.3.2 Ck/Guangxi/2461/2004 2.3.3 Ck/Guiyang/3055/2005 2.3.4Dk/Fujian/1734/2005 2.4 Ck/Yunnan/115/2004 2.5 Ck/Korea/ES/2003 2.5Ck/Korea/ES/2003(taken from Table 2 of: WHO/OIE/FAO H5N1 Evolution Working Group Emerg.Inf. Dis. 14, 7 (2008)).

The publication WHO/OIE/FAO H5N1 Evolution Working Group Emerg. Inf.Dis. 14, 7 (2008), which is incorporated herein by reference, is foundat the CDC website URL: cdc.gov/EID/content/14/7/e1.htm (12 Aug. 2011).

An overview of the clade classification of known H5N1 viruses isprovided by the phylogenetic tree at the WHO website URL:who.int/csr/disease/avian_influenza/H5CompleteTree.pdf (15 Aug. 2011),which is hereby incorporated by reference.

For determining the clade of a H5 protein of H5N1, for example, the webbased tool “Highly Pathogenic Avian Influenza (HPAI) H5N1 HA cladeprediction” can be used, which is described by Lu, Davis, Rowley, andDonis: “A Web-based tool for the clade designation of highly pathogenicavian influenza H5N1 viruses” in Options for the Control of InfluenzaVI. J. M. Katz, N. Cox & A. W. Hampson (Eds.) London: Blackwell, 2007,herein incorporated by reference, and which is found at the website URL:h5n1.flugenome.org/grouping.php (12 Aug. 2011).

For example, a H5 protein of clade 1 H5N1 virus (H5 protein (1)) is thusa HA with an amino acid sequence encoded by a nucleotide sequence of aclade 1 according to the above-mentioned WHO Nomenclature System forH5N1.

A clade 2.3.1 H5N1 virus, for instance, is hence a H5N1 falling underthe criteria of a clade 2.3.1 according to the above-mentioned WHONomenclature System for H5N1.

In a preferred embodiment, the H5 protein (1) according to theinvention, namely the H5 protein of clade 1 H5N1 virus as describedherein, comprises or consists of a polypeptide sequence having at least98.1%, preferably at least 98.2%, more preferably at least 98.3%, andmost preferably at least 98.4% sequence identity with the polypeptidesequence of SEQ ID NO:1.

Sequence identity in the context of the invention is understood as beingbased on determined pairwise similarity between protein sequences. Thedetermination of percent similarity between two sequences is preferablyaccomplished using a computational algorithm, in particular thewell-known Basic Local Alignment Search Tool (Altschul S F, Gish W,Miller W, Myers E W, Lipman D J: Basic local alignment search tool. JMol Biol 1990, 215(3):403-410). For purposes of the present invention,percent sequence identity of an amino acid sequence is determined usingthe BLAST blastp homology search algorithm using the followingparameters: an expected threshold of 10, word size 3, BLOSUM62 matrix,gap opening penalty of 11, a gap extension penalty of 1, and conditionalcompositional score matrix adjustment. The database to search against isthe set of non-redundant protein sequences (nr). The BLAST homologysearch algorithm is described in Altschul SF (1990), J Mol Biol 1990,215(3):403-410, which is herein incorporated by reference.

A variant may, for example, differ from the reference accession numberBAE07201 molecule without signal peptide (N-terminal 16 amino acidresidues are not shown in SEQ ID NO:1) by as few as 1 to 15 amino acidresidues, as few as 1 to 10 amino acid residues, such as 6-10, as few as5, as few as 4, 3, 2, or even 1 amino acid residue.

In one exemplary embodiment, the H5 protein (1) according to theinvention, i.e. the H5 protein (1) of clade 1 H5N1 virus for use in amethod of treating or preventing infections with H5N1 virus of adifferent clade, is preferably a H5 protein of influenza virus, whereinthe H5 protein having the amino acid 223N and the modification 328K+,wherein numbering of the amino acid positions of the H5 protein refersto the amino acid position as exemplarily given in SEQ ID NO:2 andwherein the modification 328K+ means that at amino acid position 328 ofH5 protein a second Lysine (K+) is inserted. Said preferred H5 protein(1) is also termed Mut k+ or mutK+ in the following. Preferably, such H5protein and any further H5 protein according to the invention is anisolated H5 protein.

The term “H5 protein (1) of clade 1 H5N1”, as used herein, preferablymeans “H5 protein (1) as single antigen of clade 1 H5N1 virus” or inparticular “H5 protein (1) as single antigen”.

The terms “hemagglutinin 5 (H5)” or “H5 of avian influenza virus” or “H5protein” as used herein are equivalent and mean, but are not limited toany naturally occurring H5 protein and any modified forms of H5 protein,including any deletion, substitution and/or insertion mutant of H5protein.

The numbering of the amino acid positions of the H5 protein (1) Mut k+as used herein refers to the amino acid position as exemplarily given inSEQ ID NO:2. SEQ ID NO:2 represents the amino sequence of thehemagglutinin of strain duck/China/E319-2/03 but lacking the aminoterminal signal peptide. In other words, if reference is made to theamino acid at position 223 (amino acid 223), the amino acid residue ismeant which corresponds to amino acid 223 of SEQ ID NO:2. However, thisdoes not mean that the H5 protein Mut k+ according to the invention hasthe identical amino acid sequence with SEQ ID NO:2. It only says, thatthe corresponding amino acids of the H5 proteins according to theinventions code for the amino acid residue, as explicitly mentioned. Inthe current case, amino acid 223 would be Serine (S). The terms “223N”,or “155N” exemplarily mean, that the amino acid at positions 223 and155, respectively—numbering according to the amino acid positions of SEQID NO:2—,that shall code for the amino acid Asparagine (N). In otherwords, if reference is made to “H5 protein (1) having the amino acid223N”, a H5 amino acid molecule that normally codes for Serine at aminoacid position 223—numbering according to the amino acid positions of SEQID NO:2—that amino acid shall be substituted by an Asparagine (N). Theterm “328K+” or “modification 328K+” means, that at amino acid position328 of H5 protein—numbering according to the amino acid positions of SEQID NO:2—, a second Lysine (K+) is inserted. In cases were amino acidssequences at positions 328 and 329 naturally codes for Lysine-Lysine, nofurther Lysine (K) shall be inserted. However, most of the known H5sequences code at amino acid positions 328 and 329 for Lysine-Arginine.In any such cases, the term 328K+ modification means, that a secondLysine (K) shall be inserted between Lysine at position 328 and Arginineat position 329. The modified sequence would read thenLysine-Lysine-Arginine (KKR).

Regarding the present example, the hemagglutinin of strainduck/China/E319-2/03 is shifted to a H5 protein (1) of clade 1 H5N1,since it resembles the H5 sequence of the clade 1 H5N1 virusA/HongKong/213/2003, the year/location/host of this HK isolate, andshows reactivity with clade-1-specific antibodies. Hence the Mut K+sequence is classified as a H5 sequence of a clade 1 H5N1. Within thecontext of the invention, the designed Mut K+ sequence is thusunderstood and defined to be a H5 protein of clade 1 H5N1 virus.

Thus, in particular also any designed H5 protein is understood anddefined as a H5 protein of clade 1 H5N1 virus according to theinvention, if it is encoded by a nucleotide sequence which fulfils thecriteria of a nucleotide sequence of a clade 1 according to theabove-mentioned WHO Nomenclature System for H5N1.

Thus, in one embodiment, the present invention is implemented with a H5protein and any modified forms of H5 protein, including any deletion,substitution and/or insertion mutant of H5 protein, wherein those H5proteins having the amino acid 223N and the modification 328K+, whereinnumbering of the amino acid positions of the H5 protein refers to theamino acid position as exemplarily given in SEQ ID NO:2 and wherein themodification 328K+ means that at amino acid position 328 of H5 protein asecond Lysine (K+) is inserted. It is self-explanatory, that any of theH5 proteins as provided herewith are antigenic, which mean they showantigenic properties in an standard hemagglutinin inhibition assay forinfluenza viruses.

According to a further embodiment, the present invention also relates toany part of the H5 protein (1), which means any peptide-fragment whichshows antigenic properties in an standard hemagglutinin inhibitionassay, having in one embodiment at least the amino acid 223N and themodification 328K+, wherein numbering of the amino acid positions of theH5 protein refers to the amino acid position as exemplarily given in SEQID NO:2 and wherein the modification 328K+ means that at amino acidposition 328 of H5 protein a second Lysine (K+) is inserted.

A H5 protein (1) shows antigenic properties if it inhibitshemagglutination in a standard hemagglutinin inhibition assay, forexample, as described in Example 2. Normally said antigenic part of H5protein (1) comprises 200, 180, 160, 150, 140, 130, 120, 110 or mostpreferably 105 contiguous amino acids of the amino acid sequence thatcodes for the H5 protein as mentioned above, modified or non-modified,which shows antigenic properties in an standard hemagglutinin inhibitionassay as described in Example 2. A standard hemagglutinin inhibitionassay for example is also described in Stephenson et al., Virus Researchvol. 103, pp. 91-95 (2004) with further references. However, the HIassay as described in Example 2 shall be understood to be the relevantreference assay in connection with all aspects of the invention asdescribed herein:

Briefly, HI assay was performed to detect the presence of HA-specificantibodies. A heterologous H5N2 virus, A/chicken/Mexico/232/94, was usedat a concentration of four hemagglutinating units [4 HA units] in the HIassay. In U-bottomed microtiter plates serial two-fold serum dilutionsin PBS were subsequently mixed with equal volumes (25 μL) containing 4HA units of virus, and incubated at room temperature (about 25° C.) for30 min. Chicken red blood cells, at a concentration of 0.5% in PBS, wereadded to the serum-virus containing wells and incubated for 40 min atroom temperature. The HI titers were determined as reciprocals of thehighest serum dilutions in which inhibition of hemagglutination wasobserved.

Of note, Haesebrouck and Pensaert (1986) found “that there may exist acorrelation between the HI titers against the challenge virus andprotection from challenge”. Haesebrouck and Pensaert (1986) alsodetermined that pigs with HI titers of ≧40 were “completely resistant tochallenge and no replication of the virus occurred in the respiratorytract at challenge”. Thus, the development of HI titers ≧40 in thevaccinated swine would correlate to protection. (F. Haesebrouck and M.B. Pensaert, 1986). Effect of intratracheal challenge of fattening pigspreviously immunized with an inactivated influenza H1N1 vaccine(Veterinary Microbiology, 11 (1986) 239-249. It has to assume thatequivalent or at least nearly equivalent H5 HI titers will also resultin a complete immune protection of swine against avian influenza virus.Lower titers, at least result in a seroconversion of the vaccinatedanimals and result in partial immune protection of those animals, whichalso can dramatically reduce the risk of a pandemics.

Moreover, an antigenic part of the H5 protein (1) according to theinvention includes, but is not limited to deletion mutants of H5protein, which comprises:

-   -   i. at least 35, 30, 25, 20, 18, 15, 13, 10, 9, or most        preferably 8 contiguous amino acids of the amino acid sequence        that surrounds and includes the amino acid 223N; and    -   ii. at least 35, 30, 25, 20, 18, 15, 13, 10, 9, or most        preferably 8 contiguous amino acids of the amino acid sequence        that surrounds and includes the amino acid modification 328K+,        and    -   iii. wherein any of such antigenic part of H5 protein shows        hemagglutinin inhibition in a standard hemagglutinin inhibition        assay as described in Example 2.

Preferably, those surrounding amino acids of amino acid 223N and/or328K+ are encoded by SEQ ID NO:2 or SEQ ID NO:5.

Furthermore preferred H5 proteins (1) according to the invention are:

-   -   i. any of those mentioned above having the amino acid 223N and        the modification 328K+;    -   ii. any of those mentioned above having the amino acid 94N/223N        and the modification 328K+;    -   iii. any H5 protein of avian origin having the amino acid 223N,        and the modification 328K+, wherein avian origin means that the        H5 sequence derived form a virus isolate that was originally        isolated from a poultry infected with avian influenza virus type        5; or    -   iv. any H5 protein of avian origin having the amino acids        94N/223N and the modification 328K+, wherein avian origin means        that the H5 sequence derived from a virus isolate that was        originally isolated from poultry infected with avian influenza        virus type 5; or    -   v. any H5 protein of avian origin having the amino acids        155N/223N and the modification 328K+, wherein avian origin means        that the H5 sequence derived from a virus isolate that was        originally isolated from poultry infected with avian influenza        virus type 5; or    -   vi. any H5 protein of avian origin having the amino acid        120N/155N/223N and the modification 328K+, wherein avian origin        means that the H5 sequence derived from a virus isolate that was        originally isolated from poultry infected with avian influenza        virus type 5; or    -   vii. any H5 protein having the modifications 94N/223N and the        modification 328K+; or    -   viii. any H5 protein having the modifications 94N/155N/223N and        the modification 328K+; or;    -   ix. any H5 protein having the modifications 94N/120N/155N/223N        and the modification 328K+; or    -   x. any H5 protein having the modifications 223N, the        modification 328K+, and one or more of the following amino acid        clusters selected from the group consisting of:        -   a. aa 93-95: GNF        -   b. aa 123-125: SDH        -   c. aa 128-130: SSG        -   d. aa 138-140: GSS        -   e. aa 226-228: MDF        -   f. aa 270-272: EVE        -   g. aa 309-311: NKL; or    -   xi. any H5 protein having the amino acid 223N, and the        modification 328K+, and one or more of the following amino acid        clusters selected from the group consisting of:        -   a. aa 93-95: GNF        -   b. aa 128-130: SSG        -   c. aa 138-140: GSS; or    -   xii. any H5 protein having the amino acid sequence of SEQ ID        NO:5.

Furthermore preferred H5 proteins (1) as provided herewith include theH5 proteins as described by Hoffmann et al, PNAS, vol. 106, no. 36, pp.12915-12920 of Sep. 6, 2005, wherein that H5 proteins includes one ormore of the modifications as described above, at least the amino acid223N and the modification 328K+, wherein numbering of the amino acidpositions of the H5 protein refers to the amino acid position asexemplarily given in SEQ ID NO:2 and wherein the modification 328K+means that at amino acid position 328 of H5 protein a second Lysine (K+)is inserted. The disclosure of this reference shall be entirely includedherein by reference.

Furthermore preferred H5 proteins (1) as provided herewith include H5proteins which comprise a peptide that comprises the amino acid 223N andthe modification 328K+, wherein numbering of the amino acid positions ofthe H5 protein refers to the amino acid position as exemplarily given inSEQ ID NO:2 and wherein the modification 328K+ means that at amino acidposition 328 of H5 protein a second Lysine (K+) is inserted, and:

-   -   i. the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID        NO:4; SEQ ID NO:5; SEQ ID NO:6 or SEQ ID NO:7 or,    -   ii. any peptide that has at least 85% sequence homology, more        preferably at least about 90% sequence homology, still more        preferably at least about 95% sequence homology, even more        preferably at least about 97% sequence homology, still even more        preferably at least about 98% sequence homology, and even more        preferably at least about 99% sequence homology to the        polypeptide of i) that comprises hemagglutinin inhibition in a        standard hemagglutinin inhibition as described above; or    -   iii. any antigenic part of the polypeptides of i) or ii)        comprising at least 35, 30, 25, 20, 18, 15, 13, 10, 9, or most        preferably 8 contiguous amino acids of any of peptides of i) or        ii).    -   iv. any peptides of i), ii) or iii) having the amino acids 36T,        36K, 83A, 83T, 83D, 86A, 86V, 120N, 120S, 155N, 155S, 156A,        156T, 189R, 189K, 212K, 212R, 212E, 223N, 223N, or 120N/155N.    -   v. any peptide of i), ii), iii) or iv) having one or more of the        following amino acid clusters selected from the group consisting        of:        -   a. aa 93-95: GNF        -   b. aa 123-125: SDH        -   c. aa 128-130: SSG        -   d. aa 138-140: GSS        -   e. aa 226-228: MDF        -   f. aa 270-272: EVE        -   g. aa 309-311: NKL; or    -   vi. any peptide of i), ii) iii) or iv) having one or more of the        following amino acid clusters selected from the group consisting        of:        -   a. aa 93-95: GNF        -   b. aa 128-130: SSG        -   c. aa 138-140: GSS.

“Sequence homology”, as used herein, refers to a method of determiningthe relatedness of two sequences. To determine sequence homology, two ormore sequences are optimally aligned, and gaps are introduced ifnecessary. In contrast to sequence identity, conservative amino acidsubstitutions are counted as a match when determining sequence homology.In other words, to obtain a polypeptide or polynucleotide having 95%sequence homology with a reference sequence, 85%, preferably 90%, evenmore preferably 95% of the amino acid residues or nucleotides in thereference sequence must match or comprise a conservative substitutionwith another amino acid or nucleotide, or a number of amino acids ornucleotides up to 15%, preferably up to 10%, even more preferably up to5% of the total amino acid residues or nucleotides, not includingconservative substitutions, in the reference sequence may be insertedinto the reference sequence. Preferably the homolog sequence comprisesat least a stretch of 50, even more preferred of 100, even morepreferred of 250, even more preferred of 500 nucleotides. Upon suchalignment, sequence homology is ascertained on a position-by-positionbasis, e.g., the sequences are “homolog” at a particular position if atthat position, the nucleotides or amino acid residues are identical. Thetotal number of such position identities is then divided by the totalnumber of nucleotides or amino acid residues in the reference sequenceto give % sequence homology. Sequence homology can be readily calculatedby known methods, including but not limited to, those described inComputational Molecular Biology, Lesk, A. N., ed., Oxford UniversityPress, New York (1988), Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey (1994); Sequence Analysis in Molecular Biology, vonHeinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov,M. and Devereux, J., eds., M. Stockton Press, New York (1991); andCarillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), theteachings of which are incorporated herein by reference. Preferredmethods to determine the sequence homology are designed to give thelargest match between the sequences tested. Methods to determinesequence homology are codified in publicly available computer programswhich determine sequence identity between given sequences. Examples ofsuch programs include, but are not limited to, the GCG program package(Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)),BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol.,215:403-410 (1990). The BLASTX program is publicly available from NCBIand other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIHBethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol.,215:403-410 (1990), the teachings of which are incorporated herein byreference). These programs optimally align sequences using default gapweights in order to produce the highest level of sequence homologybetween the given and reference sequences.

Furthermore preferred H5 proteins (1) include H5 proteins which comprisethe 328K+ modification as mentioned above, and the amino acid sequenceprovided in TABLE 1, or any immunogenic part thereof:

TABLE 1 H5 antigens Basic- Amino acid positions^(#) Sequence namesequence 36 83 86 120  155  156  189 212 223 263 223N/328K+ any HA H5 —— — — — — — — N — 36T/223N/328K+ any HA H5 T — — — — — — — N —36K/223N/328k+ any HA H5 K — — — — — — — N — 83A/223N/328k+ any HA H5 —A — — — — — — N — 83T/223N/328k+ any HA H5 — T — — — — — — N —83D/223N/328k+ any HA H5 — D — — — — — — N — 86A/223N/328k+ any HA H5 —— A — — — — — N — 86V/223N/328k+ any HA H5 — — V — — — — — N —120N/223N/328k+ any HA H5 — — — N — — — — N — 120S/223N/328k+ any HA H5— — — S — — — — N — 155N/223N/328k+ any HA H5 — — — — N — — — N —155S/223N/328k+ any HA H5 — — — — S — — — N — 156A/223N/328k+ any HA H5— — — — — A — — N — 156T/223N/328k+ any HA H5 — — — — — T — — N —189R/223N/328k+ any HA H5 — — — — — — R — N — 189K/223N/328k+ any HA H5— — — — — — K — N — 212K/223N/328k+ any HA H5 — — — — — — — K N —212R/223N/328k+ any HA H5 — — — — — — — R N — 212E/223N/328k+ any HA H5— — — — — — — E N — 223N/263A/328k+ any HA H5 — — — — — — — — N A223N/263T/328k+ any HA H5 — — — — — — — — N T 120N/155N/223N/ any HA H5— — — N N — — — N — 328k+ A/duck/China/E319- AAR99628 T A A S D A R K NA 2/03/328k+ A/duck/China/E319- AAR99628 T A A S D A R K N A2/03_223N/328k+ A/duck/China/E319- AAR99628 T A A N D A R K N A2/03_120N/223N/ 328k+ A/duck/China/E319- AAR99628 T A A S N A R K N A2/03_155N/223N/ 328k+ A/duck/China/E319- AAR99628 T A A S N N R K N A2/03_120N/155N/ 223N/328k+ HA/HK/213/03/328k+ AY518362 T A A N N A R K NA HA/Vietnam/1203/04 K T V S S T K R N T HA/Vietnam/1203/ K T V S S T KR N T 04_223N/328k+ HA/Vietnam/3046/ T A V S S T K R N T 04_223N/328k+HA/Vietnam/3062/ T A V S S T K R N T 04_223N/328k+ HA/chicken/Vietnam/ TA V S S T K R N T 39/04_223N/328k+ HA/falcon/HK- T A A S S A K E N AD0028/04_223N/328k+ HA/duck/Singapore/ T D V S N A K E N A3/97_223N/328k+ HA/HK/156/97/328k+ T A A S S A K E N T ^(#)the aminoacid positions given in TABLE 1 refers to the positions as exemplarilydefined in SEQ ID NO:2. In other words amino acid 223 of TABLE 1 refersto the amino acid 223 of the sequence of SEQ ID NO:2. — means that theamino acids at this positions are variable as compared to the referencesequence.

Furthermore, the present invention also relates to H5 proteins (1)having at least the amino acid 223N and the modification 328K+, whereinnumbering of the amino acid positions of the H5 protein refers to theamino acid position as exemplarily given in SEQ ID NO:2 and wherein themodification 328K+ means that at amino acid position 328 of H5 protein asecond Lysine (K+) is inserted, and comprises:

-   -   i. a peptide having the sequences of NCBI Accession No.        AAT65209, CAJ32556, ABC47656, CAF21874, CAF21870, AAC58998,        AAC58997, AAC58996, AAC58994, AAC58993, AAC58992, AAC58991,        AAC58990, AAC58995, AAS45134, AAN17270, AAN17269, AAN17268,        AAN17267, AAN17266, AAN17265, AAN17264, AAN17263, AAN17262,        AAN17261, AAN17260, AAN17259, AAN17257, AAN17256, AAN17255,        AAN17254, AAA43083, AAA43082, AAB19079, BAE48696, BAE48693,        BAE48696, BAE48695, BAE48694, BAE48692, BAE48691, BAE48690,        BAE48689, BAE48688, BAE48687, BAE48686, BAE48685, BAE48684,        BAE48683, AAC58999, ABC72082, AAV91149, AAP71993, AAP71992,        AAP71991, AAP71990, AAP71989, AAP72011, AAP72010, AAP72009,        AAP72008, AAP72007, AAP72006, AAP72005, AAP72004, AAP72003,        AAP72002, AAP72001, AAP72000, AAP71999, AAP71998, AAP71997,        AAP71996, AAP71995, AAP71994, AAF99718, ABF58847, AAG38534,        AAC32102, AAC32099, AAL75847, AAC32101, AAC32098, AAC32088,        AAC32078, AAR99628, AAC32100, AAM49555, AAL75843, AAL75839,        AAD13573, AAD13568, AAF04720, AAF04719, AAC34263, AAR16155,        AAD13574, AAD13570, AAD13575, AAD13572, AAD13569, AAD13567,        AAD13566, AAK57506, AAG01225, AAG01215, AAG01205, AAG01195, or        ABD83813 modified in a manner described above, which means that        those sequences include the above-mentioned modifications 223N        and 328 K+ which are not part of the wild-type sequences; or    -   ii. any peptide that has at least 85% sequence homology, more        preferably at least about 90% sequence homology, still more        preferably at least about 95% sequence homology, even more        preferably at least about 97% sequence homology, still even more        preferably at least about 98% sequence homology, and even more        preferably at least about 99% sequence homology to the        polypeptide of i) and that show hemagglutinin inhibition in a        standard hemagglutinin inhibition as described above;    -   iii. any of the peptides of i) or ii) having the amino acids        36T, 36K, 83A, 83T, 83D, 86A, 86V, 120N, 120S, 155N, 155S, 156A,        156T, 189R, 189K, 212K, 212R, 212E, 263A, 263T, or 120N/155N; or    -   iv. any of such peptides of i), ii), or iii) having one or more        of the following amino acid clusters selected from the group        consisting of:        -   a. aa 93-95:GNF        -   b. aa 123-125 SDH        -   c. aa 128-130: SSG        -   d. aa 138-140:GSS        -   e. aa 226-228: MDF        -   f. as 270-272: EVE        -   g. aa 309-311: NKL; or    -   v. any peptide of i), ii) iii) or iv) having one or more of the        following amino acid clusters selected from the group consisting        of:        -   a. as 93-95:GNF        -   b. aa 128-130: SSG        -   c. aa 138-140:GSS

Preferably, the H5 protein (1) for use in a method of treating orpreventing infections with H5N1 virus of a different clade isrecombinantly expressed and/or produced by a baculovirus expressionsystem, preferably in cultured insect cells.

The term “H5 protein (1)” as mentioned herein is thus, in particular,equivalent to the term “recombinant H5 protein” used herein.

Regarding the H5N1 virus of a different clade, as mentioned herein, saidH5N1 virus of a different clade is preferably selected from the groupconsisting of clade 0 H5N1 virus, clade 2 H5N1 virus, clade 3 H5N1virus, clade 4 H5N1 virus, clade 5 H5N1 virus, clade 6 H5N1 virus, clade7 H5N1 virus, clade 8 H5N1 virus and clade 9 H5N1 virus.

In a further preferred embodiment of the invention, the H5N1 virus of adifferent clade is clade 2.2 H5N1 virus or a clade 2.3 H5N1 virus.

In a particular preferred embodiment of the invention, the H5N1 virus ofa different clade is a clade 2.2.1 H5N1 virus or a clade 2.3.2 H5N1virus.

For reasons of clarity, H5 protein of the H5N1 virus of a differentclade is termed “H5 protein (2)” hereinafter. Hence, H5 protein (2) asmentioned herein is in particular a H5 protein coded by the genome of aH5N1 of any clade with the exception of clade 1.

In still a further preferred embodiment, the H5N1 virus of a differentclade is a H5N1 virus of North African or of Vietnamese origin, whereinsaid H5N1 virus of North African origin is preferably a H5N1 viruscomprising a H5 protein (2) of influenza virus,

wherein said H5 protein (2) has

-   -   (a) the amino acids 113D, 126H, 145(−), 156R, 160F, 167T, and        181N, wherein the modification 145(−) means that amino acid        position 145 of H5 is deleted, or    -   (b) the amino acids 87P, 145L, 172T, 201E, 206I, 208K, 254T,        341G and 421K, or    -   (c) the amino acids 145L, 172T, and 254V,    -   and wherein the numbering of the amino acid positions of the H5        protein (2) refers to the amino acid position as exemplarily        given in SEQ ID NO:8;    -   or wherein said H5 protein (2) consists of or comprises an amino        acid sequence which is at least 95%, preferably at least 96%,        more preferably at least 97%, still more preferably at least        98%, yet more preferably at least 99%, or in particular        preferred 100% homolog with any one of the sequences as set        forth in SEQ ID NOs: 9 to 46.

In the context of the invention, said H5 protein (2) according to (a) isa Subclade A protein, and said H5 protein according to (b) or (c) is aSubclade B protein.

Within the context of the invention, it is understood that the term“amino acid” in particular refers to an amino acid residue or,respectively, to an amino acid which has been covalently linked viapeptide bonds to two further amino acids or, if the amino acid is N- orC-terminally located in the peptide sequence, to one further amino acid.

In a still more preferred embodiment of the invention, the H5N1 virus ofa different clade comprises H5 protein (2) having

-   -   (a) the amino acids 87L, 113D, 126H, 145(−), 156R, 160F, 167T,        and 181N, or    -   (b) the amino acids 87P, 113N, 126R, 145L, 160Y, 172T, 181H,        201E, 206I, 208K, 254T, 341G and 421K, or    -   (c) the amino acids 87L, 113N, 126R, 145L, 156G, 160Y, 172T,        181H, and 254V,    -   and/or        wherein such H5 protein (2) comprises a peptide that comprises:    -   i. any one of the amino acid sequences of SEQ ID NOs: 9 to 46;    -   ii. any peptide that has at least 85%, preferably at least 95%,        even more preferably at least 96%, even more preferably at least        97%, even more preferably at least 98%, even more preferably at        least 99%, most preferably 100% sequence homology to the        polypeptide of i) and that comprises hemagglutinin inhibition in        a standard hemagglutinin inhibition assay; or    -   iii. any part of the polypeptides of i) or ii) comprising at        least 334 contiguous amino acids of any of such peptides of i)        or ii) and wherein any of such peptide comprises hemagglutinin        inhibition in a standard hemagglutinin inhibition assay,    -   and/or

-   wherein such H5 protein (2) consists of or comprises a contiguos    amino acid sequence which has at least 95% even more preferably at    least 96%, even more preferably at least 97%, even more preferably    at least 98%, even more preferably at least 99%, most preferably    100% sequence identity with any one of the sequences as set forth in    SEQ ID NOs: 9 to 46.

More particular, the H5N1 virus of a different clade preferablycomprises H5 protein (2) which consists of or comprises an amino acidsequence which is at least 95%, preferably at least 96%, more preferablyat least 97%, still more preferably at least 98%, yet more preferably atleast 99%, or in particular preferred 100% homolog with any one of thesequences as set forth in SEQ ID NOs: 15 or 20, and wherein such H5protein (2) comprising or consisting of the amino acid sequence setforth in SEQ ID NO:20 are in particular more preferred.

In particular, the present invention is directed to the H5 protein (1)described herein for use in a method of treating or preventinginfections

-   -   (A) with Subclade A H5N1 virus of North African origin, namely        an infection with a H5N1 virus comprising a H5 protein (2)        having the amino acids according to (a) of claim 13 or 14 or        comprising a H5 protein according to claim 16 or 17 relating to        any one of the sequences as set forth in SEQ ID NOs: 9 to 19, or        42 or 43,    -   or    -   (B) with Subclade B H5N1 virus of North African origin, namely        an infection with a H5N1 virus comprising a H5 protein having        the amino acids according to (b) or (c) of claim 13 or 14 or        comprising a H5 protein according to claim 16 or 17 relating to        any one of the sequences as set forth in SEQ ID NOs: 20 to 41,        or 44 to 46.

According to a further embodiment, the present invention also relates tonucleic acid molecules, which code for any of the H5 proteins (1), asdescribed supra, for use in a method of treating or preventinginfections with H5N1 virus of a different clade. Preferably, thosenucleic acid molecules are RNA, DNA or copy (c)DNA molecules. Thus, thepresent invention relates to a nucleic acid molecule, preferably a cDNAmolecule coding for a H5 protein or any modified forms of H5 protein,including any deletion, substitution and/or insertion mutant of H5protein, wherein those H5 proteins having the amino acid 223N and themodification 328K+, wherein numbering of the amino acid positions of theH5 protein refers to the amino acid position as exemplarily given in SEQID NO:2 and wherein the modification 328K+ means that at amino acidposition 328 of H5 protein a second Lysine (K+) is inserted.

According to a further embodiment, the present invention also relates toa nucleic acid molecule, preferably a cDNA molecule coding for any partof the H5 protein (1), which means encoding for any peptide-fragmentwhich shows antigenic properties in an standard hemagglutinin inhibitionassay as described supra, and having at least the amino acid 223N andthe modification 328K+, wherein numbering of the amino acid positions ofthe H5 protein refers to the amino acid position as exemplarily given inSEQ ID NO:2 and wherein the modification 328K+ means that at amino acidposition 328 of H5 protein a second Lysine (K+) is inserted. Normallysuch nucleic acid molecules, which code for an antigenic part of H5protein, comprise 600, 540, 480, 450, 420, 390, 360, 330 or mostpreferably 315 contiguous nucleotides of the nucleotide sequence thatcodes for the H5 protein as mentioned above, modified or non-modified,and which shows antigenic properties in an standard hemagglutinininhibition assay as described herein.

Further embodiments of antigenic parts of the H5 protein (1) aredescribed supra. It is in the common knowledge of a person skilled inthe art to construct any such nucleic acid molecules, preferably cDNAmolecules which codes for the antigenic part of the H5 protein asdescribed supra. This also include but is not limited to theconstruction of nucleic acid molecules, preferably of cDNA molecules,which codes for antigenic parts of the H5 protein as mentioned aboveincluding deletion mutants of H5 protein, which comprises:

-   -   i. at least 105, 90, 75, 60, 48, 45, 39, 30, 27, or most        preferably 24 contiguous amino nucleotides of the nucleotide        sequence that surrounds and includes the coding sequence that        codes for amino acid 223N; and    -   ii. at least 105, 90, 75, 60, 48, 45, 39, 30, 27, or most        preferably 24 contiguous amino nucleotides of the nucleotide        sequence that surrounds and includes the coding sequence that        codes for modification 328K+, and    -   iii. wherein any of such antigenic part of H5 protein show        hemagglutinin inhibition in a standard hemagglutinin inhibition        assay as described in Example 2.

Preferably, those surrounding nucleotides of the nucleotides, which codefor amino acids 223N and/or 328K+, coding for SEQ ID NO:2 or SEQ IDNO:5.

Furthermore preferred nucleic acid molecules encoding for the H5 protein(1) according to the invention are:

-   -   i. any of those mentioned supra encoding for the amino acid 223N        and the modification 328K+;    -   ii. any of those mentioned supra encoding for the amino acid        94N/223N and the modification 328K+;    -   iii. any nucleic acid molecules of avian origin encoding for the        amino acid 223N, and the modification 328K+, wherein avian        origin means that the H5 sequence derived from a virus isolate        that was originally isolated from poultry infected with avian        influenza virus type 5; or    -   iv. any nucleic acid molecules of avian origin encoding for the        amino acids 94N/223N and the modification 328K+, wherein avian        origin means that the H5 sequence derived from a virus isolate        that was originally isolated from poultry infected with avian        influenza virus type 5; or.    -   v. any nucleic acid molecules of avian origin encoding for the        amino acids 155N/223N and the modification 328K+, wherein avian        origin means that the H5 sequence derived from a virus isolate        that was originally isolated from poultry infected with avian        influenza virus type 5; or    -   vi. any nucleic acid molecule encoding for H5 protein of avian        origin having the amino acid 120N/155N/223N and the modification        328K+, wherein avian origin means that the H5 sequence derived        from a virus isolate that was originally isolated from poultry        infected with avian influenza virus type 5; or    -   vii. any nucleic acid molecule encoding for H5 protein having        the modifications 94N/223N and the modification 328K+; or    -   viii. any nucleic acid molecule encoding for H5 protein having        the modifications 94N/155N/223N and the modification 328K+; or;    -   ix. any nucleic acid molecule encoding for H5 protein having the        modifications 94N/120N/155N/223N and the modification 328K+; or    -   x. any nucleic acid molecule encoding for H5 protein having the        modifications 223N, the modification 328K+, and one or more of        the following amino acid clusters selected from the group        consisting of:        -   a. aa 93-95: GNF        -   b. aa 123-125: SDH        -   c. aa 128-130: SSG        -   d. aa 138-140: GSS        -   e. as 226-228: MDF        -   f. aa 270-272: EVE        -   g. aa 309-311: NKL; or    -   xi. any nucleic acid molecule encoding for H5 protein having the        amino acid 223N, the modification 328K+, and one or more of the        following amino acid clusters selected from the group consisting        of:        -   a. as 93-95: GNF        -   b. aa 128-130: SSG        -   c. aa 138-140: GSS; or    -   xii. any nucleic acid molecule encoding for H5 protein having        the amino acid sequence of SEQ ID NO:5.

Furthermore preferred H5 proteins (1) as provided herewith include theH5 proteins as described by Hoffmann et al, PNAS, vol. 106, no. 36, pp.12915-12920 of Sep. 6, 2005, wherein that H5 proteins includes one ormore of the modifications as described above, at least the amino acid223N and the modification 328K+, wherein numbering of the amino acidpositions of the H5 protein refers to the amino acid position asexemplarily given in SEQ ID NO:2 and wherein the modification 328K+means that at amino acid position 328 of H5 protein a second Lysine (K+)is inserted. The disclosure of this reference shall be entirely includedherein by reference. Thus according to a further embodiments, thepresent invention also relates to any nucleic acid molecule, preferablya cDNA molecule coding for any of such proteins described by Hoffmann etal, PNAS, vol. 106, no. 36, pp. 12915-12920 of Sep. 6, 2005, whereinthat H5 proteins includes one or more of the modifications as describedabove, at least the amino acid 223N and the modification 328K+, whereinnumbering of the amino acid positions of the H5 protein refers to theamino acid position as exemplarily given in SEQ ID NO:2 and wherein themodification 328K+ means that at amino acid position 328 of H5 protein asecond Lysine (K+) is inserted.

Methods, of how to introduce any of the above-mentioned modificationswithin the nucleotide sequence, including the encoding sequence of theH5 protein of an influenza virus, are well known in the art. The genomicsequence of the entire influenza virus can be modified according to theinvention, for example according to the methods described in U.S. Pat.No. 6,951,754, with further references.

Furthermore, there may be employed conventional molecular biology,microbiology, and recombinant DNA techniques within the skill of the artto modify a nucleic acid sequence coding for an antigen as describedherein. Such techniques are explained fully in the literature. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glovered. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization [B. D. Hames & S. J. Higgins eds. (1985)]; TranscriptionAnd Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal CellCulture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRLPress, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning(1984); F. M. Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, Inc. 1994).

According to a further embodiment, the present invention also relates toa vector that comprises any of such nucleic acid molecules as describedsupra. In other words, the present invention relates to a vector, thatincludes the coding sequence of any such H5 protein (1), or part thereofas described supra. Preferably, said vector is an expression vector,which allows the expression of any such H5 protein (1) or part thereofas described supra. Vectors according to the invention are those whichare suitable for the transfection or infection of bacterial, yeast oranimal cells, in vitro or in vivo.

Vectors and methods for making and/or using vectors (or recombinants)for expression can be by or analogous to the methods disclosed in: U.S.Pat. Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683,5,494,807, 4,722,848, 5,942,235, 5,364,773, 5,762,938, 5,770,212,5,942,235, 382,425, PCT publications WO 94/16716, WO 96/39491, WO95/30018, Paoletti, “Applications of pox virus vectors to vaccination:An update, “PNAS USA 93: 11349-11353, October 1996, Moss, “Geneticallyengineered poxviruses for recombinant gene expression, vaccination, andsafety,” PNAS USA 93: 11341-11348, October 1996, Smith et al., U.S. Pat.No. 4,745,051, (recombinant baculovirus), Richardson, C. D. (Editor),Methods in Molecular Biology 39, “Baculovirus Expression Protocols”(1995 Humana Press Inc.), Smith et al., “Production of Human BetaInterferon in Insect Cells Infected with a Baculovirus ExpressionVector”, Molecular and Cellular Biology, December, 1983, Vol. 3, No. 12,p. 2156-2165; Pennock et al., “Strong and Regulated Expression ofEscherichia coli B-Galactosidase in Infect Cells with a Baculovirusvector, “Molecular and Cellular Biology March 1984, Vol. 4, No. 3, p.399-406; EPA0 370 573, U.S. application Ser. No. 920,197, filed Oct. 16,1986, EP Patent publication No. 265785, U.S. Pat. No. 4,769,331(recombinant herpesvirus), Roizman, “The function of herpes simplexvirus genes: A primer for genetic engineering of novel vectors,” PNASUSA 93:11307-11312, October 1996, Andreansky et al., “The application ofgenetically engineered herpes simplex viruses to the treatment ofexperimental brain tumors,” PNAS USA 93: 11313-11318, October 1996,Robertson et al. “Epstein-Barr virus vectors for gene delivery to Blymphocytes”, PNAS USA 93: 11334-11340, October 1996, Frolov et al.,“Alphavirus-based expression vectors: Strategies and applications,” PNASUSA 93: 11371-11377, October 1996, Kitson et al., J. Virol. 65,3068-3075, 1991; U.S. Pat. Nos. 5,591,439, 5,552,143, WO 98/00166,allowed U.S. application Ser. Nos. 08/675,556, and 08/675,566 both filedJul. 3, 1996 (recombinant adenovirus), Grunhaus et al., 1992,“Adenovirus as cloning vectors,” Seminars in Virology (Vol. 3) p.237-52, 1993, Ballay et al. EMBO Journal, vol. 4, p. 3861-65, Graham,Tibtech 8, 85-87, April, 1990, Prevec et al., J. Gen Virol. 70,42434,PCT WO 91/11525, Feigner et al. (1994), J. Biol. Chem. 269, 2550-2561,Science, 259: 1745-49, 1993 and McClements et al., “Immunization withDNA vaccines encoding glycoprotein D or glycoprotein B, alone or incombination, induces protective immunity in animal models of herpessimplex virus-2 disease”, PNAS USA 93: 11414-11420, October 1996, andU.S. Pat. Nos. 5,591,639, 5,589,466, and 5,580,859, as well as WO90/11092, WO93/19183, WO94/21797, WO95/11307, WO95/20660, Tang et al.,Nature and Furth et al. Analytical Biochemistry, relating to DNAexpression vectors, inter alia. See also WO 98/33510; Ju et al.,Diabetologia, 41: 736-739, 1998 (lentiviral expression system); Sanfordet al., U.S. Pat. No. 4,945,050; Fischbach et al. (Intracel), WO90/01543; Robinson et al., seminars in Immunology vol. 9, pp. 271-283(1997), (DNA vector systems); Szoka et al., U.S. Pat. No. ______ (methodof inserting DNA into living cells); McCormick et al., U.S. Pat. No.5,677,178 (use of cytopathic viruses); and U.S. Pat. No. 5,928,913(vectors for gene delivery), as well as other documents cited herein,each of which is incorporated by reference herein.

A viral vector, for instance, selected from pig herpes viruses, such asAujeszky's disease virus, porcine adenovirus, poxviruses, especiallyvaccinia virus, avipox virus, canarypox virus, and swinepox virus, aswell as DNA vectors (DNA plasmids) are advantageously employed in thepractice of the invention.

Methods of Producing the H5 Proteins (1) According to the PresentInvention

According to another aspect, the present invention provides methods ofproducing and/or recovering high amounts of recombinant H5 protein: i)by permitting infection of susceptible cells in culture with arecombinant viral vector containing H5 DNA coding sequences, wherein H5protein is expressed by the recombinant viral vector, and ii) thereafterrecovering the H5 protein from cell culture. High amounts of H5 proteinmeans, but are not limited to, more than about 20 μg/mL cell culture,preferably more than about μg/mL, even more preferred more than about 30μg/mL, even more preferred more than about 40 μg/mL, even more preferredmore than about 50 μg/mL, even more preferred more than about 60 μg/mL,even more preferred more than about 80 μg/mL, even more preferred morethan about 100 μg/mL, even more preferred than about 150 μg/mL, mostpreferred more than about 190 μg/mL.

According to a preferred embodiment, the H5 protein (1) is recovered byharvesting the whole (i.e. intact) SF+ cells expressing the H5 protein.

Preferred cells are those susceptible for infection with an appropriaterecombinant viral vector, containing a H5 DNA and expressing the H5protein (1). Preferably the cells are insect cells, and more preferably,they include the insect cells sold under the trademark SF+ insect cells(Protein Sciences Corporation, Meriden, Conn.). Preferred cell cultureshave a cell count between about 0.3−2.0×10⁶ cells/mL, more preferablyfrom about 0.35−1.9×10⁶ cells/mL, still more preferably from about0.4−1.8×10⁶ cells/mL, even more preferably from about 0.45−1.7×10⁶cells/mL, and most preferably from about 0.5−1.5×10⁶ cells/mL.

Preferred viral vectors include baculovirus such as BaculoGold (BDBiosciences Pharmingen, San Diego, Calif.), in particular provided thatthe production cells are insect cells. Although the baculovirusexpression system is preferred, it is understood by those of skill inthe art that other expression systems will work for purposes of thepresent invention, namely the expression of H5 into the supernatant of acell culture. Such other expression systems may require the use of asignal sequence in order to cause H5 expression into the media.

Appropriate growth media will also be determinable by those of skill inthe art with a preferred growth media being serum-free insect cell mediasuch as Excell 420 (JRH Biosciences, Inc., Lenexa, Kans.) and the like.

The recombinant viral vector containing the H5 DNA sequences has apreferred multiplicity of infection (MOI) of between about 0.03-1.5,more preferably from about 0.05-1.3, still more preferably from about0.09-1.1, and most preferably from about 0.1-1.0, when used for theinfection of the susceptible cells. Preferably the MOls mentioned aboverelates to one mL of cell culture fluid. Preferably, the methoddescribed herein comprises the infection of 0.35−1.9×10⁶ cells/mL, stillmore preferably of about 0.4−1.8×10⁶ cells/mL, even more preferably ofabout 0.45−1.7×10⁶ cells/mL, and most preferably of about 0.5−1.5×10⁶cells/mL with a recombinant viral vector containing a H5 DNA andexpressing the H5 protein having a MOI (multiplicity of infection) ofbetween about 0.03-1.5, more preferably from about 0.05-1.3, still morepreferably from about 0.09-1.1, and most preferably from about 0.1-1.0.

The infected cells are then incubated over a period of up to ten days,more preferably from about two days to about ten days, still morepreferably from about four days to about nine days, and most preferablyfrom about five days to about eight days. Preferred incubationconditions include a temperature between about 22-32° C., morepreferably from about 24-30° C., still more preferably from about 25-29°C., even more preferably from about 26-28° C., and most preferably about27° C. Preferably, the SF+ cells are observed following inoculation forcharacteristic baculovirus-induced changes. Such observation may includemonitoring cell density trends and the decrease in viability during thepost-infection period. It was found that peak viral titer is observed3-5 days after infection and peak H5 protein expression in the cells isobtained between days 5 and 8, and/or when cell viability decreases toless than 10%.

Thus, one aspect of the present invention provides a method of producingand/or recovering recombinant H5 protein, preferably in amountsdescribed above, by i) permitting infection of a number of susceptiblecells (see above) in culture with a recombinant viral vector with a MOIas defined above, ii) expressing H5 protein by the recombinant viralvector, and iii) thereafter recovering the H5 protein from the cellsobtained between days 5 and 8 after infection and/or cell viabilitydecreases to less then 10%. Preferably, the recombinant viral vector isa recombinant baculovirus containing H5 DNA coding sequences and thecells are SF+ cells. Additionally, it is preferred that the culture beperiodically examined for macroscopic and microscopic evidence ofcontamination or for atypical changes in cell morphology during thepost-infection period. Any culture exhibiting any contamination shouldbe discarded.

For recovery of H5 protein (1) that will be used in an immunogenic orimmunological composition such as a vaccine, the inclusion of aninactivation step is preferred in order to inactivate the viral vector.

An “immunogenic or immunological composition” refers to a composition ofmatter that comprises at least one antigen which elicits animmunological response in the host of a cellular and/orantibody-mediated immune response to the composition or vaccine ofinterest. Usually, an “immunological response” includes but is notlimited to one or more of the following effects: the production oractivation of antibodies, B cells, helper T cells, suppressor T cells,and/or cytotoxic T cells and/or gamma-delta T cells, directedspecifically to an antigen or antigens included in the composition orvaccine of interest. Preferably, the host will display either atherapeutic or protective immunological response such that resistance tonew infection will be enhanced and/or the clinical severity of thedisease reduced. Such protection will be demonstrated by either areduction or lack of symptoms normally displayed by an infected host, aquicker recovery time and/or a lowered viral titer in the infected host.

As used herein, “vaccine” refers to that term as it is used by those ofskill in the art. More particularly, “vaccine” refers to an immunogeniccomposition that, when administered to an animal in need thereof,results in a reduction in the incidence of or severity of clinical signsof influenza infection up to an including the complete prevention ofsuch clinical signs. Preferably, the reduction in incidence or severityis at least 10%, more preferably at least 20%, still more preferably atleast 30%, even more preferably at least 40%, more preferably at least50%, still more preferably at least 60%, even more preferably at least70%, more preferably at least 80%, still more preferably at least 90%,even more preferably at least 95%, and most preferably 100% incomparison to an animal or group of animals that did not receive thecompositions of the present invention but that were exposed toinfectious levels of influenza virus that would normally result ininfluenza infection resulting in exhibiting clinical signs.

Thus, the present invention also relates to a method of producing and/orrecovering recombinant H5 protein, preferably in amounts describedabove, by i) permitting infection of a number of susceptible cells (seeabove) in culture with a recombinant viral vector with a MOI as definedabove, ii) expressing H5 protein by the recombinant viral vector, iii)recovering the H5 expressed in cells obtained between days 5 and 8 afterinfection and/or cell viability decreases to less then 10%, and iv)inactivating the recombinant viral vector.

Preferably, this inactivation is done either just before or just afterthe filtration step, with after the filtration step being the preferredtime for inactivation. Any conventional inactivation method can be usedfor purposes of the present invention. Thus, inactivation can beperformed by chemical and/or physical treatments. In preferred forms,the volume of harvest fluids is determined and the temperature isbrought to between about 32-42° C., more preferably between about 34-40°C., and most preferably between about 35-39° C. Preferred inactivationmethods include the addition of cyclized binary ethylenimine (BEI),preferably in a concentration of about 1 to about 20 mM, preferably ofabout 2 to about 10 mM, still more preferably of about 2 to about 8 mM,still more preferably of about 3 to about 7 mM, most preferably of about5 mM. For example the inactivation includes the addition of a solutionof 2-bromoethyleneamine hydrobromide, preferably of about 0.4M, whichhas been cyclized to 0.2M binary ethylenimine (BEI) in 0.3N NaOH, to thefluids to give a final concentration of about 5 mM BEI. Preferably, thefluids are then stirred continuously for 72-96 hours and the inactivatedharvest fluids can be stored frozen at −40° C. or below or between about1-7° C. After inactivation is completed a sodium thiosulfate solution,preferably at 1.0M is added to neutralize any residual BEI. Preferably,the sodium thiosulfate is added in equivalent amount as compared to theBEI added prior to for inactivation. For example, in the event BEI isadded to a final concentration of 5 mM, a 1.0M sodium thiosulfatesolution is added to give a final minimum concentration of 5 mM toneutralize any residual BEI.

Thus, one further aspect of the present invention relates to a method ofproducing recombinant H5 protein, preferably in amounts described above,by i) permitting infection of a number of susceptible cells (see above)in culture with a recombinant viral vector with a MOI as defined above,ii) expressing H5 protein by the recombinant viral vector, iii)recovering the H5 expressed in the cells obtained between days 5 and 8after infection and/or cell viability decreases to less then 10%, andiv) inactivating the recombinant viral vector. Preferably, therecombinant viral vector is a baculovirus containing H5 DNA codingsequences and the cells are SF+ cells. Preferred inactivation steps arethose described above. Preferably, inactivation is performed betweenabout 35-39° C. and in the presence of 2 to 8 mM BEI, still morepreferred in the presence of about 5 mM BEI.

According to one further aspect of the present invention, the methoddescribed above also includes a neutralization step after step iv). Thisstep v) comprises adding of an equivalent amount of an agent thatneutralizes the inactivation agent within the solution. Preferably, ifthe inactivation agent is BEI, addition of sodium thiosulfate to anequivalent amount is preferred. Thus, according to a further aspect,step v) comprises adding of a sodium thiosulfate solution to a finalconcentration of about 1 to about 20 mM, preferably of about 2 to about10 mM, still more preferably of about 2 to about 8 mM, still morepreferably of about 3 to about 7 mM most preferably of about 5 mM, whenthe inactivation agent is BEI.

In preferred forms and especially in forms that will use the recombinantH5 protein in an immunogenic composition such as a vaccine, each lot ofharvested H5 protein will be tested for inactivation by passage in theanchorage dependent, baculovirus susceptible insect cells, such as Sf9cells. In a preferred form of this testing, 150 cm² of appropriate cellculture monolayer is inoculated with 1.0 mL of inactivated H5 fluids andmaintained at 25-29° C. for 14 days with at least two passages. At theend of the maintenance period, the cell monolayers are examined forcytopathogenic effect (CPE) typical of H5 baculovirus. Preferably,positive virus controls are also used. Such controls can consist of oneculture of Sf9 cells inoculated with a non-inactivated reference H5baculovirus and one flask of Sf9 cells that remain non-inoculated. Afterincubation and passage, the absence of virus-infected cells in the BEItreated viral fluids would constitute a satisfactory inactivation test.The control cells inoculated with the reference virus should exhibit CPEtypical of H5 baculovirus and the non-inoculated flask should notexhibit any evidence of H5 baculovirus CPE. Alternatively, at the end ofthe maintenance period, the supernatant samples could be collected andinoculated onto a Sf9 96 well plate, which has been loaded with Sf9cells, and then maintained at 25-29° C. for 5-6 days. The plate is thenfixed and stained with anti-H5 antibody conjugated to FITC or anylabeled antibody directed to baculovirus specific proteins (i.e. gp64).The absence of CPE, H5 expression, or expression of baculovirus specificproteins (i.e. gp64) in the BEI treated viral fluids constitutes asatisfactory inactivation test. The control cells inoculated with thereference virus should exhibit CPE and IFA activity and thenon-inoculated flask should not exhibit any evidence of H5 baculovirusCPE and contain no IFA activity.

Thus a further aspect described herein, relates to an inactivation testfor determining the effectiveness of the inactivation of therecombination viral vector expressing H5 protein (1), comprises thesteps: i) contacting at least a portion of the culture fluid containingthe recombinant viral vector with an inactivating agent, preferably asdescribed above, ii) adding a neutralization agent to neutralize theinactivation agent, preferably as described above, and iii) determiningthe residual infectivity by the assays as described above.

After inactivation, the relative amount of recombinant H5 protein in asample can be determined in a number of ways. Preferred methods ofquantitation include SDS-PAGE densitometry, ELISA, and animalvaccination studies that correlate known quantities of vaccine withclinical outcomes (serology, etc.). When SDS-PAGE is utilized forquantitation, the sample material containing an unknown amount ofrecombinant H5 protein is run on a gel, together with samples thatcontain different known amounts of recombinant H5 protein. A standardcurve can then be produced based on the known samples and the amount ofrecombinant H5 in the unknown sample can be determined by comparisonwith this standard curve. Because ELISAs are generally recognized as theindustry standard for antigen quantitation, they are preferred forquantitation.

Vaccines Comprising H5 Proteins (1 or Nucleic Acid Molecules or VectorsCoding for Those

The invention further provides a combination of

-   (a) the H5 protein (1) described herein    and-   (b) an inactivated Newcastle disease virus    for use in a method of treating or preventing infections with H5N1    virus of a different clade, in particular for use in any method of    treating or preventing infections with H5N1 virus of a different    clade as described herein.

Said combination is also termed “the combination described herein”hereinafter.

According to the invention it is understood that the combinationdescribed herein is preferably included in a multivalent combinationvaccine or the combination described herein is in particular directed toa combined vaccination, more particular to an administration of the H5protein (1) described herein and of the inactivted Newcastle diseasevirus within a maximum of 24 hours to an animal, in particular poultry,or human being in need thereof.

Preferably, the inactivated Newcastle disease virus is an inactivatedwhole Newcastle disease virion.

In another preferred embodiment, the inactivated Newcastle disease virusis an inactivated Newcastle disease virus obtained by inactivation of aNewcastle disease virus comprising a RNA polynucleotide having at least70%, preferably at least 80%, more preferably at least 90%, still morepreferably at least 95% or in particular 100% sequence identity with aRNA copy of the polynucleotide set forth in SEQ ID NO: 51 (cDNA sequenceof LaSota strain virus), which has been inactivated.

In particular, the inactivated Newcastle disease virus is an inactivatedNewcastle disease LaSota strain virus.

In one preferred embodiment the inactivated Newcastle Disease Virus is aNewcastle Disease Virus which has been inactivated with a reagentselected from the group consisting of Formaldehyde, binary ethyleneimine(BEI), Beta-Propio-Lactone (BPL), and combinations thereof.

The amount of inactivated Newcastle disease virus in the combinationdescribed herein is preferably between 10² and 10¹⁰ equivalents of egginfectious doses (EID50), preferably between 10⁶ and 10⁹ EID50, inparticular preferably between 10⁷ and 10⁹ EID50. The amount of the H5protein (1) in the combination described herein is preferably the sameas mentioned hereinafter.

The amount of the H5 protein (1) according to the invention ispreferably between 10 and 1000 Hemagglutination units (HAU's) per dose,more preferably between 50 and 950 HAU's per dose, even more preferablybetween 100 and 900 HAU's per dose, even more preferably between 200 and800 HAU's per dose, even more preferably between 300 and 700 HAU's perdose, still more preferably between 300 and 500 HAU's per dose.

According to a further aspect, the present invention relates to vaccinesor pharmaceutical compositions in general, that comprises,

-   -   i. one or more of the H5 proteins (1) as described herein or the        combination described herein;    -   ii. one or more of the nucleic acid molecules as described        herein, coding for any such H5 proteins (1); and/or    -   iii. one or more of the vectors as described herein, including        any such nucleic acid molecules and coding for any such H5        proteins (1) as described herein; and    -   iv. a pharmaceutical acceptable carrier and/or excipient.

The term “pharmaceutical composition” “Pharmaceutical/vaccinecomposition” as described herein, includes but is not limited to,vaccines for the reduction or prevention of an infection or to acomposition of matter for the treatment and lessening of an infection.

The preparation of nucleic acid based vaccines, preferably cDNAvaccines, coding for influenza hemagglutinin are described for examplein Deck et al, Vaccine 1997; 15(1):71-78; Ulmer et al., Science 1993;259:1745-1749; Ulmer et al., Vaccine 1994; 12(16):1541-1544. Any ofthose methods can be used for the production of nucleic acid basedvaccines, preferably cDNA vaccines, coding for an influenza H5 proteinas described herein.

Moreover, a vaccine, which comprises H5 protein (1) or parts thereof asdescribed herein, can be produced by conventional approaches, e.g. byrecombinant expression techniques or by biochemical purification andseparation techniques. Recombinant expression techniques, including theexpression in insect cells are well known in the art, and described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II (D. N.Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984);Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)];Transcription And Translation [B. D. Hames & S. J. Higgins, eds.(1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; ImmobilizedCells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide ToMolecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, Inc. 1994). Further examples ofwell established recombinant expression systems are bacterial expressionsystems such as E. coli or B. subtilis, yeast-based expression systemssuch as S. cerevisiae or S. pombe, or mammalian cell expression systemssuch as the BHK-, CHO- and/or NSO-based expression systems. Such systemsare well known in the art and generally available, e.g. commerciallythrough Clontech Laboratories, Inc. 4030 Fabian Way, Palo Alto, Calif.94303-4607, USA. Further expression strategies are for example describedin Lüschow et al., Vaccine no. 19 (2001), pp. 4249-4259, or Veit et al.,PNAS vol. 103 (2006), pp. 8197-8202. Furthermore, recombinantadeno-associated virus systems are well established and for exampledescribed in U.S. Pat. No. 5,436,146 or WO200203872 with furtherreferences. Moreover, vaccinia (pox) virus based expression systems, forexample as described in U.S. Pat. No. 6,265,183 with further references,are also well established and suitable to produce recombinantantigen(s), antigenic composition(s) as used according to the invention.Further suitable expression systems make use of recombinant popovaviruses, such as SV40, fowl pox virus, pseudorabies viruses andretroviruses.

The relevant pharmaceutical/vaccine compositions as described herein,can also comprise inactivated virus which comprises H5 protein (1) asdescribed herein, an apathogenic version of a live virus comprising H5protein (1) as described herein, preparation and/or fragments of avirus, wherein said preparation and/or fragment comprise the H5 protein(1) as described herein.

The skilled person knows additional components which may be comprised insaid compositions/vaccines together with antigen (see for example,Remington's Pharmaceutical Sciences. (1990). 18th ed. Mack Publ.,Easton). The expert may use known injectable, physiologically acceptablesterile solutions. For preparing a ready-to-use solution, aqueousisotonic solutions, such as e.g. saline or corresponding plasma proteinsolutions, are readily available. The pharmaceutical composition/vaccinemay be present as lyophylisates or dry preparations, which can bereconstituted with a known injectable solution directly before use understerile conditions, e.g. as a kit of parts.

In addition the pharmaceutical/vaccine compositions of the presentinvention can include one or more veterinary-acceptable carriers. Asused herein, “a veterinary-acceptable carrier” includes but is notlimited to any and all solvents, dispersion media, coatings, adjuvants,stabilizing agents, diluents, preservatives, antibacterial andantifungal agents, isotonic agents, adsorption delaying agents, and thelike.

Diluents can include water, saline, dextrose, ethanol, glycerol, and thelike. Isotonic agents can include sodium chloride, dextrose, mannitol,sorbitol, and lactose, among others. Stabilizers include albumin andalkali salts of ethylendiamintetracetic acid, among others.

A preservative as used herein, refers to an anti-microbiological activeagent, such as for example Gentamycin, Merthiolate, and the like. Inparticular adding of a preservative is most preferred for thepreparation of a multi-dose composition. Those anti-microbiologicalactive agents are added in concentrations effective to prevent thecomposition of interest for any microbiological contamination or forinhibition of any microbiological growth within the composition ofinterest.

“Adjuvants” as used herein, can include aluminum hydroxide and aluminumphosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc.,Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham,Ala.), water-in-oil emulsion, oil-in-water emulsion,water-in-oil-in-water emulsion.

The emulsion can be based in particular on light liquid paraffin oil(European Pharmacopoeia type); isoprenoid oil such as squalane orsqualene; oil resulting from the oligomerization of alkenes, inparticular of isobutene or decene; esters of acids or of alcoholscontaining a linear alkyl group, more particularly plant oils, ethyloleate, propylene glycol di-(caprylate/caprate), glyceryltri-(caprylate/caprate) or propylene glycol dioleate; esters of branchedfatty acids or alcohols, in particular isostearic acid esters. The oilis used in combination with emulsifiers to form the emulsion. Theemulsifiers are preferably nonionic surfactants, in particular esters ofsorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, ofpolyglycerol, of propylene glycol and of oleic, isostearic, ricinoleicor hydroxystearic acid, which are optionally ethoxylated, andpolyoxypropylene-polyoxyethylene copolymer blocks, in particular thePluronic products, especially L121. See Hunter et al., The Theory andPractical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.). JohnWiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570(1997). Examples for suitable oil-in water emulsions are Emulsigen-basedadjuvants, such as EMULSIGEN®, EMULSIGEN-D®, EMULSIGEN-P®, EMULSIGEN-75®(MVP Laboratories, Inc. Omaha, Nebr., USA). It has been surprisinglyfound, that pharmaceutical/vaccine compositions that comprise H5protein, preferably recombinant H5 protein as described herein, havebeen effectively adjuvanted with oil-in water emulsions, preferably withsuch Emulsigen-based adjuvants, more preferably with EMULSIGEN® andEMULSIGEN-D®.

Moreover, it is possible to use the SPT emulsion described on page 147of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M.Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59described on page 183 of this same book.

A further instance of an adjuvant is a compound chosen from the polymersof acrylic or methacrylic acid and the copolymers of maleic anhydrideand alkenyl derivative. Advantageous adjuvant compounds are the polymersof acrylic or methacrylic acid which are cross-linked, especially withpolyalkenyl ethers of sugars orpolyalcohols. These compounds are knownby the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Personsskilled in the art can also refer to U.S. Pat. No. 2,909,462 whichdescribes such acrylic polymers cross-linked with a polyhydroxylatedcompound having at least 3 hydroxyl groups, preferably not more than 8,the hydrogen atoms of at least three hydroxyls being replaced byunsaturated aliphatic radicals having at least 2 carbon atoms. Thepreferred radicals are those containing from 2 to 4 carbon atoms, e.g.vinyls, allyls and other ethylenically unsaturated groups. Theunsaturated radicals may themselves contain other substituents, such asmethyl. The products sold under the name Carbopol; (BF Goodrich, Ohio,USA) are particularly appropriate. They are cross-linked with an allylsucrose or with allyl pentaerythritol. Among then, there may bementioned Carbopol 974P, 934P and 971P. Most preferred is the use ofCarbopol 971P. Among the copolymers of maleic anhydride and alkenylderivative, the copolymers EMA (Monsanto) which are copolymers of maleicanhydride and ethylene. The dissolution of these polymers in water leadsto an acid solution that will be neutralized, preferably tophysiological pH, in order to give the adjuvant solution into which theimmunogenic, immunological or vaccine composition itself will beincorporated.

Further suitable adjuvants include, but are not limited to, the RIBIadjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.),SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridinelipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinantor otherwise), cholera toxin, or muramyl dipeptide among many others.

Preferably, the adjuvant is added in an amount of about 100 μg to about10 mg per dose. Even more preferred the adjuvant is added in an amountof about 100 μg to about 10 mg per dose. Even more preferred theadjuvant is added in an amount of about 500 μg to about 5 mg per dose.Even more preferred the adjuvant is added in an amount of about 750 μgto about 2.5 mg per dose. Most preferred the adjuvant is added in anamount of about 1 mg per dose.

The pharmaceutical/vaccine compositions, can further include one or moreother immunomodulatory agents such as, e.g., interleukins, interferons,or other cytokines. The pharmaceutical/vaccine compositions can alsoinclude Gentamicin and Merthiolate. While the amounts and concentrationsof adjuvants and additives useful in the context of the presentinvention can readily be determined by the skilled artisan, the presentinvention contemplates compositions comprising from about 50 μg to about2000 μg of adjuvant and preferably about 250 ug/1 ml dose of the vaccinecomposition. In another preferred embodiment, the present inventioncontemplates vaccine compositions comprising from about 1 ug/ml to about60 μg/ml of antibiotics, and more preferably less than about 30 μg/ml ofantibiotics.

Thus, according to a further embodiment, the present invention alsorelates to a pharmaceutical/vaccine composition comprising

-   -   i. a therapeutically effective amount of any one of the H5        proteins of influenza virus as described herein, wherein the H5        protein having the amino acid 223N and the modification 328K+,        wherein numbering of the amino acid positions of the H5 protein        refers to the amino acid position as exemplarily given in SEQ ID        NO:2 and wherein the modification 328K+ means that at amino acid        position 328 of H5 protein a second Lysine (K+) is inserted; and    -   ii. a pharmaceutically acceptable adjuvants as described above.

Preferably, the adjuvant is selected from the group consisting of:

-   -   a) EMULSIGEN®, a oil-in-water emulsion (o/w);    -   b) EMULSIGEN-D®, a oil-in-water (o/w) with        dimethyldioctadecylammonum bromide (DDA);    -   c) a Polygen, a copolymer    -   d) EMULSIGEN-P®, a oil-in-water (o/w) with a proprietary        immunostimulant    -   e) Carbigen is a cross-linked polymer    -   f) EMULSIGEN-75®, a double adjuvants comprise of a oil-in-water        (o/w) with a cross-linked polymer    -   g) ISA 70 is a water-in-oil (w/o)

Most preferably, the adjuvants is a oil-in-water emulsion such as anemulsigen-based adjuvant selected from the group consisting ofEMULSIGEN®, EMULSIGEN-D®, EMULSIGEN-P®, EMULSIGEN-75®, EMULSIGEN® andEMULSIGEN-P®. Most preferably EMULSIGEN® and EMULSIGEN-P® are used inthe formulation of the current invention.

According to a further aspect, the pharmaceutical/vaccine compositionsas provided herewith, comprise one or more antigen. Preferably, thatfurther antigen is an antigen of a poultry or mammalian pathogen.According to a further embodiments, that additional antigen is anfurther influenza antigen such as hemagglutinin H5, H7, H9, or any otherhemagglutinin of influenza virus, wherein the H5 is preferably a H5protein of a H5N1 virus of a clade different than clade 1, in particularof a H5N1 virus of North African origin, such as the H5 protein (2)described herein. The additional antigen(s) can be added in a purifiedform, as part of an antigenic preparation, in the form of a killedmicroorganism or in the form of a modified live microorganism.

The term “antigen”, as used herein means, but is not limited to,peptides, polypeptides, glycopeptides, or polysaccharides which arecapable of specifically interacting with an antigen recognition moleculeof the immune system, such as an immunoglobulin (antibody) or T cellantigen receptor in order to elicit, activate or stimulate an immuneresponse directed to said antigen in a host to which said antigen isadministered. The term “antigen” also refers to nucleic acid molecules,preferably DNA- or RNA-molecules, each of which codes for and express apeptide, polypeptide, or glycopeptide that is capable of specificallyinteracting with an antigen recognition molecule of the immune system,such as an immunoglobulin (antibody) or T cell antigen receptor in orderto elicit, activate or stimulate an immune response against the antigenthat is coded by the nucleic acid molecule. The antigen used for thepreparation of the pharmaceutical composition which is used according tothe invention is a microorganism or an antigenic part and/or preparationof said microorganism. In this connection, the term “immunization”, asused herein, means but is not limited to, any cause or enhancement of animmune response. The term “immune response” is already described supra.

Administration strategies for influenza vaccines are well known in theart. Mucosal vaccination strategies for inactivated and attenuated virusvaccines are contemplated. While the mucosa can be targeted by localdelivery of a vaccine, various strategies have been employed to deliverimmunogenic proteins to the mucosa.

In a specific embodiment, the vaccine can be administered in anadmixture with, or as a conjugate or chimeric fusion protein with,cholera toxin, such as cholera toxin B or a cholera toxin A/B chimera(Hajishengallis, J Immunol., 154:4322-32, 1995; Jobling and Holmes,Infect Immun., 60:4915-24, 1992). Mucosal vaccines based on use of thecholera toxin B subunit have been described (Lebens and Holmgren, DevBiol Stand 82:215-27, 1994). In another embodiment, an admixture withheat labile enterotoxin (LT) can be prepared for mucosal vaccination.

Other mucosal immunization strategies include encapsulating the virus inmicrocapsules (U.S. Pat. No. 5,075,109, U.S. Pat. No. 5,820,883, andU.S. Pat. No. 5,853,763) and using an immunopotentiating membranouscarrier (WO 98/0558). Immunogenicity of orally administered immunogenscan be enhanced by using red blood cells (rbc) or rbc ghosts (U.S. Pat.No. 5,643,577), or by using blue tongue antigen (U.S. Pat. No.5,690,938).

According to another aspect, the present invention relates to a methodfor preparing a pharmaceutical/vaccine composition as described above,preferably a method for producing a vaccine which comprises arecombinant, baculovirus expressed H5 protein as described supra.Generally, this method includes the steps of transfecting a constructinto a virus, wherein the construct comprises i) recombinant H5 cDNA asdescribed herein, ii) infecting cells in growth media with thetransfected virus, iii) causing the virus to express the recombinant H5protein as described herein iv) recovering the expressed H5 protein fromthe culture v) and preparing the composition by blending the expressedH5 protein with a suitable adjuvant and/or other pharmaceuticallyacceptable carrier.

Preferred adjuvants are those described above. Thus according to afurther aspect, the method for preparing an antigenic composition, suchas for example a vaccine, for invoking an immune response againstinfluenza infections comprises i) preparing and recovering H5 protein,and ii) admixing this with a suitable adjuvants.

In addition, the vaccine composition of the present invention can alsoinclude diluents, isotonic agents, stabilizers, an/or preservatives.Diluents can include water, saline, dextrose, ethanol, glycerol, and thelike. Isotonic agents can include anorganic or organic salts, e.g.sodium chloride, dextrose, mannitol, sorbitol, and lactose, saccharides,trehalose, mannitol, saccharose among others. Stabilizers includealbumin and alkali salts of ethylendiamintetracetic acid, among others.Suitable adjuvants, are those described above.

Medicinal Use of any of Such H5 Proteins (1), Nucleic Acid Molecules,Vectors, Vaccines, and Combinations Described Herein

The H5 proteins (1) as provided herewith, the nucleic acid moleculescoding for any such H5 proteins (1), the vectors comprising any suchnucleic acid molecules coding for any such H5 proteins (1) as describedherein, and any pharmaceutical/vaccine composition comprising any ofsuch H5 protein (1), nucleic acid molecule or vector or the combinationdescribed herein can be used as a medicine, preferably for the treatmentand prophylaxis of infections, caused by influenza virus, mostpreferably by influenza A virus. The H5 proteins (1) as providedherewith, the nucleic acid molecules encoding for any such H5 proteins,the vectors comprising any such nucleic acid molecules encoding for anysuch H5 proteins (1) as described herein, and any pharmaceutical/vaccinecomposition comprising any of such H5 protein (1), nucleic acid moleculeor vector, as described herein, or the combination described herein canbe used for the treatment or prophylaxis of human beings as well as inveterinary medicine. When used in veterinary medicine, the treatment ofpoultry, preferably bird, chicken, duck, turkey and the like as well asmammals, preferably pigs, cattle, horses, seals, camels, dogs, cats,hamsters, mice and the like, is preferred.

In terms of the present invention, “prophylaxis” refers to the reductionin the incidence of or severity of clinical signs of influenza infectionup to an including the complete prevention of such clinical signs.Preferably, the reduction in incidence or severity is at least 10%, morepreferably at least 20%, still more preferably at least 30%, even morepreferably at least 40%, more preferably at least 50%, still morepreferably at least 60%, even more preferably at least 70%, morepreferably at least 80%, still more preferably at least 90%, even morepreferably at least 95%, and most preferably 100% in comparison to ananimal or group of animals that did not receive the compositions of thepresent invention but that were exposed to infectious levels ofinfluenza virus that would normally result in influenza infectionresulting in exhibiting clinical signs.

Thus, according to another aspect the present invention relates to theuse of H5 proteins (1) as provided herewith, the nucleic acid moleculesencoding for any such H5 proteins (1), the vectors comprising any suchnucleic acid molecules encoding for any such H5 proteins (1) asdescribed herein and any pharmaceutical/vaccine compositions comprisingany of such H5 protein (1), nucleic acid molecule or vector as describedherein or the combination described herein, can be used as a medicine,preferably as a medicine for human beings and/or as veterinary medicine,preferably for poultry, in particular for chicken.

Moreover, H5 proteins (1) as provided herewith, the nucleic acidmolecules coding for any such H5 proteins (1), the vectors comprisingany such nucleic acid molecules coding for any such H5 protein (1), asdescribed herein, or the combination described herein can be used forthe preparation of a pharmaceutical composition, as described herein,preferably of a single-shot vaccine or a one dose vaccine, for theprophylaxis or treatment of infections caused by H5N1 virus of a cladeother than clade 1, wherein said H5N1 virus of a clade other than clade1 is preferably the H5N1 virus of a different clade as described herein.As mentioned above, those pharmaceutical compositions/vaccinecompositions can be used for the treatment and/or prophylaxis of humanbeings as well as for the treatment and/or prophylaxis of animals, suchas poultry, preferably bird, chicken, duck, turkey and the like as wellas mammals, preferably pigs, cattle, horses, seals, camels, dogs, cats,hamsters, mice and the like.

According to a further aspect, the present invention also relates to amethod for the treatment or prophylaxis of influenza virus infectionscaused by H5N1 virus of a clade other than clade 1, wherein said H5N1virus of a clade other than clade 1 is preferably the H5N1 virus of adifferent clade as described herein, wherein the method comprisingadministration of a therapeutically effective amount of the H5 protein(1) as described herein or of the combination described herein, to asubject in need of such a treatment. Moreover, the present inventionalso relates to a method for the treatment or prophylaxis of influenzavirus infections caused by H5N1 virus of a clade other than clade 1,wherein said H5N1 virus of a clade other than clade 1 is preferably theH5N1 virus of a different clade as described herein, wherein the methodcomprising administration of a therapeutically effective amount of anyH5 nucleic acid molecule or vector as described herein, that codes forany H5 protein (1) as described herein, to a subject in need of such atreatment. Furthermore, the present invention also relates to a methodfor the treatment or prophylaxis of influenza virus infections caused byH5N1 virus of a clade other than clade 1, wherein said H5N1 virus of aclade other than clade 1 is preferably the H5N1 virus of a differentclade described herein, wherein the method comprising administration ofa therapeutically effective amount of the vaccine comprising any such H5protein (1), nucleic acid molecule or vector, as described herein, to asubject in need of such a treatment. The subject in need thereof can bea human being as well as an animal, preferably poultry, even morepreferably bird, chicken, duck, turkey or a mammal, preferably pig,cattle, horse, seal, camel, dog, cat, hamster, mouse and the like.

Preferably, the administration, as described herein, is a single-shotadministration or a one dose administration.

Preferably, when chicken are vaccinated, the H5 protein as describedherein can be used for vaccination at day 1 of age or later, e.g. at day10, or at day 1 to 10, or at day 10 or later.

Preferably the influenza infection that can be treated by theadministration of any H5 protein (1), the nucleic acid molecule orvector encoding for any such H5 protein, or any pharmaceutical/vaccinecompositions as described herein, is caused by H5N1 virus of a cladeother than clade 1, wherein said H5N1 virus of a clade other than clade1 is preferably the H5N1 virus of a different clade as described hereinand, as the case may be, also in combination with another avian, swineor human influenza virus or any combination or hybrid thereof.

A further advantage of the present invention is that it benefits a‘DIVA’ (Differentiation of Infected and Vaccinated Animals) concept withspecific Elisa Kits for differentiating between vaccinated human beingsor animals and human beings or animals infected with H5N1 virus.

According to another aspect, the present invention relates to a kit ofparts, that comprises i) any of such H5 protein (1) as described herein,the nucleic acid molecule or vector encoding for any such H5 protein, orany pharmaceutical/vaccine composition comprising any of such H5protein, nucleic acid molecule or vector as described herein, and ii) apackage leaflet indicating the use of such H5 protein, nucleic acidmolecule, vector or vaccine for the treatment or prophylaxis ofinfections caused by H5N1 virus of a clade other than clade 1, whereinsaid H5N1 virus of a clade other than clade 1 is preferably the H5N1virus of a different clade as described herein. When chicken arevaccinated, the H5 protein (1) as described herein can be used forvaccination at day I on age or later.

It is thus understood that the kit of parts as mentioned herein is forthe use, or is used, respectively, for the treatment or prophylaxis ofinfections caused by H5N1 virus of a clade other than clade 1, whereinsaid H5N1 virus of a clade other than clade 1 is preferably the H5N1virus of a different clade as described herein.

According to a further embodiment, that kit in parts comprises at leastone further antigen of a poultry or mammalian pathogen and theinformation indication the medicinal, human or veterinary use of thatadditional antigen, in particular the further antigen as mentionedabove.

The invention further provides a method for reducing viral shedding in asubject, comprising administering the H5 protein (1) described herein orthe combination as described herein to a subject infected with or atrisk of a viral infection with H5N1 virus of a clade other than clade 1,wherein said H5N1 virus of a clade other than clade 1 is preferably theH5N1 virus of a different clade as described herein.

The invention also relates to the H5 protein (1) described herein or thecombination as described herein for use in a method for reducing viralshedding in a subject, wherein said H5 protein (1) or said combinationis to be administered to a subject infected with or at risk of a viralinfection with H5N1 virus of a clade other than clade 1, and whereinsaid H5N1 virus of a clade other than clade 1 is preferably the H5N1virus of a different clade as described herein.

Also, the invention provides the use of the H5 protein (1) describedherein or of the combination as described herein for the preparation ofa medicament for reducing viral shedding in a subject infected with orat risk of a viral infection with H5N1 virus of a clade other than clade1, wherein said H5N1 virus of a clade other than clade 1 is preferablythe H5N1 virus of a different clade as described herein.

Preferably, the H5 protein (1) according to the invention, thecombination described herein, the vaccine as described herein or the kitmentioned herein is for use as a single-shot vaccine or in a one-dosevaccination.

EXAMPLES

The following examples set forth preferred materials and procedures inaccordance with the present invention. It is to be understood, however,that these examples are provided by way of illustration only, andnothing therein should be deemed a limitation upon the overall scope ofthe invention.

Example 1 Construction of a Recombinant Baculoviruses Coding for andExpressing HA H5 Antigens

The recombinant baculovirus containing the H5 HA antigen was generatedas follows: the coding sequences of the H5 HA (SEQ ID NO:3) waschemically synthesized and subcloned into the transfer vector pVL1392(BD Biosciences Pharmingen, San Diego, Calif.). The H5 HA MutK+ (SEQ IDNO:5) was generated by using oligonucleotide primers and the QuikChange®Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.) andsubcloned into the transfer vector pVL1392 (BD Biosciences Pharmingen,San Diego, Calif.). The pVL1392 plasmids containing the genes coding forH5 HA antigen (SEQ ID NO:3) and H5 HA MutK+ (SEQ ID NO:5) were thenco-transfected with DiamondBac® (Sigma) baculovirus DNA into Sf9 insectcells (BD Biosciences Pharmingen) to generate the recombinantbaculovirus containing the genes H5 HA coding for SEQ ID NO:3 and H5 HAmutK+ coding for SEQ ID NO:5. The recombinant baculoviruses containingthe genes coding for H5 HA (SEQ ID NO:3) and H5 HA MutK+ (SEQ ID NO:5)were plaque-purified and Master Seed Viruses (MSVs) were propagated onthe SF+ cell line, aliquoted, and stored at −70° C. Insect cellsinfected with H5 HA baculoviruses As described above to generate MSV orWorking Seed Viruses express H5 HA antigen (SEQ ID NO:3) and H5 HA MutK+(SEQ ID NO:5) antigen as detected by polyclonal serum or monoclonalantibodies in an indirect fluorescent antibody assay or Western blot.

After being seeded with the appropriate amounts of recombinantbaculoviruses (H5 HA and H5 HA MutK+, respectively), spinner flaskscontaining SF+ cells (Protein Sciences, Inc., Meriden, Conn.) were thenincubated at 27±2° C. for 7 days and with stirring 100 rpm during thattime. The flasks used ventilated caps to allow for air flow. The crudewhole cell culture containing baculovirus infected SF+ cells and thecell culture supernatents of each culture were harvested.

Example 2 Preparation of Pharmaceutical Compositions (Vaccines)Comprising HA H5 Antigens

The crude whole cell H5 HA protein and H5 HA Mutk+ protein expressed ininsect cells by baculovirus-based expression system were harvested.Baculoviruses were inactivated in the presence of 5 mM cyclized binaryethylenimine (BEI) (final concentration) between about 32 and 39° C. for72 to 96 hours. After inactivation is completed, a 0.3 M sodiumthiosulfate solution was added to a final concentration of 5 mM toneutralize any residual BEI. After neutralization, various adjuvantswere added and the following vaccine/pharmaceutical compositions weregenerated.

VACCINES Generic product 501 name Antigen Crude whole-cell H5 HA proteinexpressed in insect cells by a baculovirus-based expression system.Formulation An experimental vaccine comprised of cultured insect cellsand supernatant expressing recombinant H5 HA. The vaccine was adjuvantedwith Emulsigen. Generic product 502 name Antigen Crude whole-cell H5 HAprotein expressed in insect cells by a baculovirus-based expressionsystem. Formulation An experimental vaccine comprised of cultured insectcells and supernatant expressing recombinant H5 HA. The vaccine wasadjuvanted with Emulsigen-D. Generic product 503 name Antigen Crudewhole-cell H5 HA protein expressed in insect cells by abaculovirus-based expression system. Formulation An experimental vaccinecomprised of cultured insect cells and supernatant expressingrecombinant H5 HA. The vaccine was adjuvanted with Polygen. Genericproduct 504 name Antigen Crude whole-cell H5 HA protein expressed ininsect cells by a baculovirus-based expression system. Formulation Anexperimental vaccine comprised of cultured insect cells and supernatantexpressing recombinant H5 HA. The vaccine was adjuvanted withEmulsigen-P. Generic product 505 name Antigen Crude whole-cell H5 HAprotein expressed in insect cells by a baculovirus-based expressionsystem. Formulation An experimental vaccine comprised of cultured insectcells and supernatant expressing recombinant H5 HA. The vaccine wasadjuvanted with Carbigen. Generic product 506 name Antigen Crudewhole-cell H5 HA protein expressed in insect cells by abaculovirus-based expression system. Formulation An experimental vaccinecomprised of cultured insect cells and supernatant expressingrecombinant H5 HA. The vaccine was adjuvanted with Emulsigen-75. Genericproduct 507 name Antigen Crude whole-cell H5 HA protein expressed ininsect cells by a baculovirus-based expression system. Formulation Anexperimental vaccine comprised of cultured insect cells and supernatantexpressing recombinant H5 HA. The vaccine was adjuvanted with ISA 70.Generic product 508 name Antigen Crude whole-cell H5 HA mutK+ proteinexpressed in insect cells by a baculovirus-based expression system.Formulation An experimental vaccine comprised of cultured insect cellsand supernatant expressing recombinant H5 HA. The vaccine was adjuvantedwith Emulsigen. Generic product 509 name Antigen Crude whole-cell H5 HAmutK+ protein expressed in insect cells by a baculovirus-basedexpression system. Formulation An experimental vaccine comprised ofcultured insect cells and supernatant expressing recombinant H5 HA. Thevaccine was adjuvanted with Emulsigen-D. Generic product 510 nameAntigen Crude whole-cell H5 HA mutK+ protein expressed in insect cellsby a baculovirus-based expression system. Formulation An experimentalvaccine comprised of cultured insect cells and supernatant expressingrecombinant H5 HA. The vaccine was adjuvanted with Polygen. Genericproduct 511 name Antigen Crude whole-cell H5 HA mutK+ protein expressedin insect cells by a baculovirus-based expression system. Formulation Anexperimental vaccine comprised of cultured insect cells and supernatantexpressing recombinant H5 HA. The vaccine was adjuvanted withEmulsigen-P. Generic product 512 name Antigen Crude whole-cell H5 HAmutK+ protein expressed in insect cells by a baculovirus-basedexpression system. Formulation An experimental vaccine comprised ofcultured insect cells and supernatant expressing recombinant H5 HA. Thevaccine was adjuvanted with Carbigen. Generic product 513 name AntigenCrude whole-cell H5 HA mutK+ protein expressed in insect cells by abaculovirus-based expression system. Formulation An experimental vaccinecomprised of cultured insect cells and supernatant expressingrecombinant H5 HA. The vaccine was adjuvanted with Emulsigen-75. Genericproduct 514 name Antigen Crude whole-cell H5 HA K+ protein expressed ininsect cells by a baculovirus-based expression system. Formulation Anexperimental vaccine comprised of cultured insect cells and supernatantexpressing recombinant H5 HA. The vaccine was adjuvanted with ISA 70.

Example 3 Vaccination of Chicken Against Avian Influenza

A combination vaccine comprising H5 HA Mutk+ (Fraction 1) andinactivated Newcastle disease virus (Fraction 2), named “BACULO AI+NDKV” has been evaluated in animal trials. The vaccine was formulated withthe haemmagglutinin H5 produced in the Baculovirus expression systembased on the MutK+ construct (Examples 1 and 2). The origin of theNewcastle Disease (ND) virus fraction is the whole virus.

Fraction 1:

Recombinant, baculovirus-expressed, H5 hemagglutinin (H5 HA) from AvianInfluenza H5N1 virus. Avian Influenza (AI) fraction.

AI fraction is inactivated with binary ethyleneimine (BEI). No residualinfectivity coming from Baculovirus vector is allowed.

Fraction 2:

Whole virion, Newcastle Disease Virus (ND), LaSota Strain. NewcastleDisease fraction.

ND fraction is inactivated with Formaldehyde, BEI or Beta-Propio-Lactone(BPL). No residual infectivity coming from ND virus is allowed.

Formula Composition:

Inactivated harvest material from H5 HA protein and ND are blended intoa water/oil emulsion. The mixture includes mineral oil as an adjuvant.

For evaluation of vaccine efficacy, three clinical parameters wereconsidered: 1) Morbility/mortality. 2) Antibodies levels. 3) Viralshedding.

In all studies SPF chickens were vaccinated, administration of thevaccine was by subcutaneous route, in the back of the neck. A dose of0.5 ml was administered unless otherwise stated.

Chickens were maintained inside isolator units during the whole durationof the studies. Studies were compliant with OIE international guidelinesfor evaluation of Avian Influenza vaccines.

Challenge was conducted to evaluate the Avian Influenza (AI) antigenicfraction. Chickens were inoculated 3 weeks after vaccination by theintra-nasal (50 μl) and oral (50 μl) route administering a total of 100μl of allantoic fluid containing 10⁶ EID₅₀ of the challenge virus.

To evaluate protection from challenge against HPAI H5N1 two studies wereconducted:

1) Protectotypes study, using a single or double vaccination (evaluatingboosting effect), ages of 1 day old or 10 days old chickens (evaluatingage effect), and doses of 0.5 or 0.2 ml (evaluating dose effect).

Two different challenge strains were used for this study: a) A subclade2.3.2 Vietnamese strain (isolated in 2006) which has been recentlycausing disease in South-East-Asia (China, Vietnam) Poultry production.b) A subclade 2.2.1 group B1 Egyptian strain (isolated in 2010), whichhas been recently causing disease in Egyptian Poultry production.Challenge strains are not genetically close to the vaccine baculovirusconstruct (MutK+). Results are interpreted in the context ofprotectotypes as broadening up the protection conferred for twoimmunizations with similar or different vaccines.

Conclusions:

-   -   1) Protection between 80 and 100% was observed depending on the        age or dose. 100% protection was observed when administered as        0.5 ml dose at 10 days old of the bivalent formulation.    -   2) When administered as a single 0.5 ml immunization of BACULO        AI+ND KV at 10 days of age, the same protection is observed than        administering two shots of the inactivated        traditionally-produced comercial Volvac AI KV vaccine.    -   3) When administered as a single 0.5 ml immunization of BACULO        AI+ND KV at 10 days of age, similar level of H5-specific        antibodies were detected in comparison with administering two        shots of the inactivated traditionally-produced comercial Volvac        AI KV vaccine.    -   4) Low levels of viral shedding were observed until 3 days        post-challenge, when the vaccine was administered as a single        0.5 ml immunization of BACULO AI+ND KV at 10 days of age.        2) BACULO efficacy study, using a single, unique vaccination at        10 days of age.

Three different challenge strains were used for this study: a) Asubclade 2.2.1 Egyptian strain (isolated in 2008). b) A subclade 2.2.1group A1 Egyptian strain (isolated in 2010). c) A subclade 2.2.1 groupB1 Egyptian strain (isolated in 2010). The last two have been recentlycausing disease in Egyptian Poultry production.

Conclusions:

-   -   1) Protection between 90 and 100% was observed.    -   2) Vaccine BACULO AI+ND KV showed performance compliant with        European Medicine Agency (EMA) guidelines for vaccines against        HPAI virus in birds.    -   3) This is the first report available demonstrating efficacy        with a single shot administration for a baculovirus-based        vaccine including a hemagglutinin genetically distant from those        of the viruses used for challenge.

Example 4 1. Experimental Design

This experiment was designed and conducted similar to the abovedescribed Example 3:

For evaluation of vaccine efficacy, three clinical parameters wereconsidered: 1) Morbility/mortality. 2) Antibodies levels. 3) Viralshedding.

In all studies SPF chickens were vaccinated, the administration of thevaccine was by subcutaneous route, in the back of the neck. A vaccineprototype containing a clade 1 H5 protein was used (called Mut K+)formulated as a bivalent product with a second, ND (Newcastle diseasevirus) antigenic fraction.

A dose of 0.5 ml was administered unless otherwise stated. Animals werevaccinate at 10 days of age.

Chickens were maintained inside isolator units during the whole durationof the studies. Studies were compliant with OIE international guidelinesfor evaluation of Avian Influenza vaccines.

Challenge was conducted to evaluate the Avian Influenza (AI) antigenicfraction. Chickens were inoculated 3 weeks after vaccination by theintra-nasal (50 μl) and oral (50 μl) route administering a total of 100μl of allantoic fluid containing 10⁶ EID₅₀ of the challenge virus.

This is also summarized in the table (Table A) below (Vaccination wasperformed at 10 days of age, column 1 (ID of experimental groupsaccording to the vaccine applied), column 2 (Vaccine dose), and column 3(Challenge age)).

Challenge virus was A/Chicken/Egypt/1063/2010, which is classified assubclade 2.2.1.1 HP AIV H5N1 subtype. This is the official challengestrain used in Egypt for evaluation of vaccine batches. The challengedose was 10⁶ EID₅₀.

2. Results & Data Analysis

Results & Data analysis are summarized in the table below (Table A):Column 4 (HI GMT (Geometric Mean Titre) 3 weeks post-vaccination,pre-challenge), column 5 (Percentage of survival, 2 weekspost-challenge), and column 6 (Detection of viral shedding, RT-PCRpositive samples).

TABLE A Summary of the experimental design and of the results and dataanalysis of Example 4. Experimental group Percentage (10 Challenge GMTmeasured at of Viral Sheddi

chickens dose 31 days of age survival Detection

each) Vaccine (age) Homologous Heterologous post- viral RNA us

-Vaccine Dose -Strain (vaccine (challenge challenge RT-PCR-

ID- (age) 1063- strain) virus) (%) (#positives/to

Mut K+ 0.5 ml 10⁶ 9.1 0.9 100  2/10 No vaccine (10 days EID₅₀ — — 010/10 of age) (31 days of age)

indicates data missing or illegible when filed

3. Conclusions

-   -   The vaccinated group survived the challenge. The vaccine        prototype triggered an efficient immune response, as measured as        HI titration using the homologous antigen.    -   The Mut K+ vaccine prototype provided good virological        protection, as measured as ability to reduce viral shedding.        RT-PCR Ct values were far low to represent infectious virus but        only residual genetical material instead.

In the Sequence Listing (SEQ ID NOs: 1 to 51):

SEQ ID NO: 1 corresponds to H5 of A/Hong Kong/213/2003(H5N1) withoutsignal peptide,

SEQ ID NOs: 2-7 correspond to SEQ ID NOs: 1-6 of the international (PCT)application number PCT/US2007/082699,

SEQ ID NO: 8 corresponds to H5 sequence of H5N1 “1709-6”,

SEQ ID NO: 9 corresponds to H5 sequence of H5N1 “1553-1/A1”,

SEQ ID NO: 10 corresponds to H5 sequence of H5N1 “1553-15/A1”,

SEQ ID NO: 11 corresponds to H5 sequence of H5N1 “2095-50/A1”,

SEQ ID NO: 12 corresponds to H5 sequence of H5N1 “3982-2/A1”,

SEQ ID NO: 13 corresponds to H5 sequence of H5N1 “3982-5/A1”,

SEQ ID NO: 14 corresponds to H5 sequence of H5N1 “3982-7/A1”,

SEQ ID NO: 15 corresponds to H5 sequence of H5N1 “3982-8/A1”,

SEQ ID NO: 16 corresponds to H5 sequence of H5N1 “3982-9/A1”,

SEQ ID NO: 17 corresponds to H5 sequence of H5N1 “3982-12/A1”,

SEQ ID NO: 18 corresponds to H5 sequence of H5N1 “3982-20/A1”,

SEQ ID NO: 19 corresponds to H5 sequence of H5N1 “3982-44/A1”,

SEQ ID NO: 20 corresponds to H5 sequence of H5N1 “1553-2/B1”,

SEQ ID NO: 21 corresponds to H5 sequence of H5N1 “1553-6/B1”,

SEQ ID NO: 22 corresponds to H5 sequence of H5N1 “1553-13/B2”,

SEQ ID NO: 23 corresponds to H5 sequence of H5N1 “1553-26/B2”,

SEQ ID NO: 24 corresponds to H5 sequence of H5N1 “1553-28/B1”,

SEQ ID NO: 25 corresponds to H5 sequence of H5N1 “2095-39/B2”,

SEQ ID NO: 26 corresponds to H5 sequence of H5N1 “2095-46/B1”,

SEQ ID NO: 27 corresponds to H5 sequence of H5N1 “2095-49/B1”,

SEQ ID NO: 28 corresponds to H5 sequence of H5N1 “2095-65/B1”,

SEQ ID NO: 29 corresponds to H5 sequence of H5N1 “2095-68/B2”,

SEQ ID NO: 30 corresponds to H5 sequence of H5N1 “2095-70/B2”,

SEQ ID NO: 31 corresponds to H5 sequence of H5N1 “2095-73/B2”,

SEQ ID NO: 32 corresponds to H5 sequence of H5N1 “2095-75/B2”,

SEQ ID NO: 33 corresponds to H5 sequence of H5N1 “3982-3/B1”,

SEQ ID NO: 34 corresponds to H5 sequence of H5N1 “3982-4/B1”,

SEQ ID NO: 35 corresponds to H5 sequence of H5N1 “3982-13/B1”,

SEQ ID NO: 36 corresponds to H5 sequence of H5N1 “3982-14/B2”,

SEQ ID NO: 37 corresponds to H5 sequence of H5N1 “3982-19/B3”,

SEQ ID NO: 38 corresponds to H5 sequence of H5N1 “3982-21/B2”,

SEQ ID NO: 39 corresponds to H5 sequence of H5N1 “3982-43/B1”,

SEQ ID NO: 40 corresponds to H5 sequence of H5N1 “3982-50/B1”,

SEQ ID NO: 41 corresponds to H5 sequence of H5N1 “3982-52/B1”,

SEQ ID NO: 42 corresponds to H5 sequence of H5N1 “3982-55/A1”,

SEQ ID NO: 43 corresponds to H5 sequence of H5N1 “3982-56/A1”,

SEQ ID NO: 44 corresponds to H5 sequence of H5N1 “3982-78/B2”,

SEQ ID NO: 45 corresponds to H5 sequence of H5N1 “4794-17/B”,

SEQ ID NO: 46 corresponds to H5 sequence of H5N1 “4794-18/B”,

SEQ ID NO: 47 corresponds to H5 sequence translated from SEQ ID NO: 50,

SEQ ID NO: 48 codes for a H5 sequence of H5N1 “3982-8/A1” (SEQ ID NO:15),

SEQ ID NO: 49 codes for a H5 sequence of H5N1 “1553-2/B1” (SEQ ID NO:20),

SEQ ID NO: 50 corresponds to the consensus sequence obtained afteranalysis of the 38 H5 HA gene sequences coding for SEQ ID NOs: 9 to 46,

SEQ ID NO: 51 corresponds to the cDNA of Newcastle Disease Virus LaSotastrain.

1. An H5 protein of a clade 1 H5N1 virus for use in a method of treatingor preventing infections with a H5N1 virus of a different clade, whereinsaid H5 protein comprises a polypeptide sequence having at least 98%sequence identity with the polypeptide sequence of SEQ ID NO:
 1. 2. TheH5 protein according to claim 1, wherein said H5 protein comprises apolypeptide sequence having at least 98.1%, preferably at least 98.2%,more preferably at least 98.3%, and most preferably at least 98.4%sequence identity with the polypeptide sequence of SEQ ID NO:
 1. 3. TheH5 protein according to claim 1, wherein said H5 protein has the aminoacid 223N and the modification 328K+, wherein numbering of the aminoacid positions of the H5 protein refers to the amino acid position asexemplarily given in SEQ ID NO:2, and wherein the modification 328K+means that at amino acid position 328 of H5 protein a second Lysine (K+)is inserted.
 4. The H5 protein according to claim 1, wherein such H5protein has the amino acid 94N.
 5. The H5 protein according to claim 1,wherein such H5 protein has the amino acid 120N.
 6. The H5 proteinaccording to claim 1, wherein such H5 protein has the amino acid 155N.7. The H5 protein according to claim 1, wherein such H5 protein has oneor more of the following amino acid clusters selected from the groupconsisting of: a. aa 93-95: GNF b. aa 123-125: SDH c. aa 128-130: SSG d.aa 138-140: GSS e. aa 226-228: MDF f. aa 270-272: EVE g. aa 309-311:NKL.
 8. The H5 protein according to claim 1, wherein such H5 proteincomprises a peptide comprising: i. the amino acid sequences of SEQ IDNO:5; SEQ ID NO:6 or SEQ ID NO:7; or ii. any peptide that has at least85% sequence homology to the polypeptide of i) and that compriseshemagglutinin inhibition in a standard hemagglutinin inhibition assay;or iii. any part of the polypeptides of i) or ii) comprising at least 8contiguous amino acids of any of such peptides of i) or ii) and whereinany of such peptide comprises hemagglutinin inhibition in a standardhemagglutinin inhibition assay; or iv. any peptide of i), ii) or iii)having one of the amino acids 36T, 36K, 83A, 83T, 83D, 86A, 86V, 120S,155S, 156A, 156T, 189R, 189K, 212K, 212R, 212E, 263A or 263T; or v. anypeptide of i), ii), iii) or iv) having one or more of the followingamino acid clusters selected from the group consisting of: a. aa 93-95:GNF b. aa 123-125: SDH c. aa 128-130: SSG d. aa 138-140: GSS e. aa226-228: MDF f. aa 270-272: EVE g. aa309-311: NKL.
 9. The H5 proteinaccording to claim 1, wherein such H5 protein comprises the amino acidsequence of SEQ ID NO:5.
 10. The H5 protein according to claim 1,wherein such H5 protein is recombinantly expressed and/or produced by abaculovirus expression system, preferably in cultured insect cells. 11.The H5 protein according to claim 1, wherein said H5N1 virus of adifferent clade is selected from the group consisting of clade 0 H5N1virus, clade 2 H5N1 virus, clade 3 H5N1 virus, clade 4 H5N1 virus, clade5 H5N1 virus, clade 6 H5N1 virus, clade 7 H5N1 virus, clade 8 H5N1 virusand clade 9 H5N1 virus.
 12. The H5 protein according to claim 1, whereinsaid H5N1 virus of a different clade is clade 2.2 H5N1 virus or a clade2.3 H5N1 virus.
 13. The H5 protein according to claim 1, wherein saidH5N1 virus of a different clade is a clade 2.2.1 H5N1 virus or a clade2.3.2 H5N1 virus.
 14. The H5 protein according to claim 1, wherein saidH5N1 virus of a different clade is a H5N1 virus of North African or ofVietnamese origin.
 15. The H5 protein according to claim 14, whereinsaid H5N1 virus of North African origin is a H5N1 virus comprising asecond H5 protein of influenza virus, wherein said second H5 proteinencodes an amino acid sequence including at least one member of a groupconsisting of: (a) the amino acids 113D, 126H, 145(−), 156R, 160F, 167T,and 181N, wherein the modification 145(−) means that amino acid position145 of H5 is deleted, or (b) the amino acids 87P, 145L, 172T, 201E,206I, 208K, 254T, 341G and 421K, or (c) the amino acids 145L, 172T, and254V, and wherein the numbering of the amino acid positions of saidsecond H5 protein refers to the amino acid position as exemplarily givenin SEQ ID NO:8; or wherein said second H5 protein consists of an aminoacid sequence which is at least 95%, preferably at least 96%, morepreferably at least 97%, still more preferably at least 98%, yet morepreferably at least 99%, or in particular preferred 100% homolog withany one of the sequences as set forth in SEQ ID NOs: 9 to
 46. 16. The H5protein according to claim 1, wherein said H5N1 virus of a differentclade comprising a second H5 protein encodes an amino acid sequenceincluding at least one member of the group consisting of: (a) the aminoacids 87L, 113D, 126H, 145(−), 156R, 160F, 167T, and 181N, or (b) theamino acids 87P, 113N, 126R, 145L, 160Y, 172T, 181H, 201E, 206I, 208K,254T, 341G and 421K, or (c) the amino acids 87L, 113N, 126R, 145L, 156G,160Y, 172T, 181H, and 254V, and/or wherein such second H5 proteincomprises a peptide comprising: i. any one of the amino acid sequencesof SEQ ID NOs: 9 to 46; ii. any peptide that has at least 85%,preferably at least 95%, even more preferably at least 96%, even morepreferably at least 97%, even more preferably at least 98%, even morepreferably at least 99%, most preferably 100% sequence homology to thepolypeptide of i) and that comprises hemagglutinin inhibition in astandard hemagglutinin inhibition assay; or iii. any part of thepolypeptides of i) or ii) comprising at least 334 contiguous amino acidsof any of such peptides of i) or ii) and wherein any of such peptidecomprises hemagglutinin inhibition in a standard hemagglutinininhibition assay, and/or wherein such second H5 protein comprises acontiguous amino acid sequence which has at least 95%, even morepreferably at least 96%, even more preferably at least 97%, even morepreferably at least 98%, even more preferably at least 99%, mostpreferably 100% sequence identity with any one of the sequences as setforth in SEQ ID NOs: 9 to
 46. 17. The H5 protein according to claim 1,wherein said H5N1 virus of a different clade comprising a second H5protein consists of an amino acid sequence which is at least 95%,preferably at least 96%, more preferably at least 97%, still morepreferably at least 98%, yet more preferably at least 99%, or inparticular preferred 100% homolog with any one of the sequences as setforth in SEQ ID NOs: 15 or 20, and wherein such second H5 proteinconsisting of the amino acid sequence set forth in SEQ ID NO:20 is morepreferred.
 18. The H5 protein according to claim 1, for use in a methodof treating or preventing viral infection, wherein said viral infectionincludes a virus from at least one member of a group selected from: (A)Subclade A H5N1 virus of North African origin, namely an infection witha H5N1 virus comprising a second H5 protein encoded by the amino acidsequence according to at least one of the sequences as set forth in SEQID NOs: 9 to 19, or 42 or 43, or (B) with Subclade B H5N1 virus of NorthAfrican origin, namely an infection with a H5N1 virus comprising a H5protein encoded by the amino acid sequence according to at least one ofthe sequences as set forth in SEQ ID NOs: 20 to 41, or 44 to
 46. 19. Acombination of an H5 protein of a clade 1 H5N1 virus for use in a methodof treating or preventing infections with a H5N1 virus of a differentclade, wherein said H5 protein comprises a polypeptide sequence havingat least 98% sequence identity with the polypeptide sequence of SEQ IDNO: 1 and an inactivated Newcastle disease virus.
 20. The combination ofclaim 19, wherein the inactivated Newcastle disease virus is aninactivated whole Newcastle disease virion.
 21. The combination of claim19, wherein the inactivated Newcastle disease virus is an inactivatedNewcastle disease virus obtained by inactivation of a Newcastle diseasevirus comprising a RNA polynucleotide having at least 70%, preferably atleast 80%, more preferably at least 90%, still more preferably at least95% or in particular 100% sequence identity with a RNA copy of thepolynucleotide set forth in SEQ ID NO: 51, which has been inactivated.22. The combination of claim 19, wherein the Newcastle disease virus isa Newcastle disease LaSota strain virus.
 23. The combination of claim19, wherein the Newcastle Disease Virus is inactivated with a reagentselected from the group consisting of Formaldehyde, BEI,Beta-Propio-Lactone (BPL), and combinations thereof.
 24. A vaccine foruse in a method of treating or preventing infections with a H5N1 virus,comprising: a. the H5 protein of a different clade, wherein said H5protein comprises a polypeptide sequence having at least 98% sequenceidentity with the polypeptide sequence of SEQ ID NO:1 of, and b. apharmaceutical acceptable carrier and/or excipient.
 25. The vaccineaccording to claim 24, wherein the excipient is one or more adjuvants.26. The vaccine according to claim 25, wherein the adjuvant is anEmulsigen-based adjuvant.
 27. The vaccine according to claim 24, whereinthe vaccine comprises one or more further antigens.
 28. The vaccineaccording to claim 27, wherein the one or more further antigen is anantigen of a poultry pathogen.
 29. The vaccine according to claim 28,wherein the one or more further antigen is H5, H7, or H9 of influenzavirus.
 30. The vaccine according to claim 29, wherein the H5 ofinfluenza virus is H5 protein of a H5N1 virus of a clade different thanclade
 1. 31. Use of the H5 protein wherein said H5 protein comprises apolypeptide sequence having at least 98% sequence identity with thepolypeptide sequence of SEQ ID NO:1, for the preparation of apharmaceutical composition, preferably of a single-shot vaccine or a onedose vaccine, for the prophylaxis or treatment of infections caused byH5N1 virus of a clade other than clade
 1. 32. A method for the treatmentor prophylaxis of influenza virus infections caused by H5N1 virus of aclade other than clade 1, wherein the method comprises administration ofa therapeutically effective amount of the H5 protein, wherein said H5protein comprises a polypeptide sequence having at least 98% sequenceidentity with the polypeptide sequence of SEQ ID NO: 1, to a subject inneed of such a treatment.
 33. A method for the treatment or prophylaxisof influenza virus infections caused by H5N1 virus of a clade other thanclade 1, wherein the method comprises administration of atherapeutically effective amount of a vaccine comprising: a. the H5protein of a different clade, wherein said H5 protein comprises apolypeptide sequence having at least 98% sequence identity with thepolypeptide sequence of SEQ ID NO: 1 of, and b. a pharmaceuticalacceptable carrier and/or excipient to a subject in need of such atreatment.
 34. The method of claim 32 wherein said administration is asingle-shot administration or a one dose administration.
 35. A kit ofparts, comprising: a. the H5 protein according to claim 1; and b. apackage leaflet indicating the use of such H5 protein, combination orvaccine of a) for the treatment or prophylaxis of infections caused byH5N1 virus of a clade other than clade
 1. 36. The kit according toclaims 35, wherein such kit comprises at least one or more furtherantigens of poultry or mammalian pathogen.
 37. (canceled)
 38. A methodof reducing the incidence of or severity of influenza infectioncomprising the step of administering a composition selected from thegroup consisting of: a. an H5 protein of a clade 1 H5N1 virus, whereinsaid H5 protein comprises a polypeptide sequence having at least 98%sequence identity with the polypeptide sequence of SEQ ID NO: 1, and b.a combination of the H5 protein of group a) and an inactivated Newcastledisease virus.
 39. (canceled)
 40. Use of the H5 protein wherein said H5protein comprises a polypeptide sequence having at least 98% sequenceidentity with the polypeptide sequence of SEQ ID NO:1, for thepreparation of a medicament for reducing viral shedding in a subjectinfected with or at risk of a viral infection with H5N1 virus of a cladeother than clade
 1. 41. A vaccine for use in a method of treating orpreventing infections with a H5N1 virus, comprising: a. the combinationof the H5 protein of a different clade and an inactivated Newcastledisease virus, and b. a pharmaceutical acceptable carrier and/orexcipient.
 42. The vaccine according to claim 41, wherein the excipientis one or more adjuvants.
 43. The vaccine according to claim 41, whereinthe adjuvant is an Emulsigen-based adjuvant.
 44. The vaccine accordingto claim 41, wherein the vaccine comprises one or more further antigens.45. The vaccine according to claim 44, wherein the one or more furtherantigen is an antigen of a poultry pathogen.
 46. The vaccine accordingto claim 45, wherein the one or more further antigen is H5, H7, or H9 ofinfluenza virus.
 47. The vaccine according to claim 46, wherein the H5of influenza virus is H5 protein of a H5N1 virus of a clade differentthan clade
 1. 48. Use of the combination of the H5 protein wherein saidH5 protein comprises a polypeptide sequence having at least 98% sequenceidentity with the polypeptide sequence of SEQ ID NO:1 and an inactivatedNewcastle disease virus, for the preparation of a pharmaceuticalcomposition, preferably of a single-shot vaccine or a one dose vaccine,for the prophylaxis or treatment of infections caused by H5N1 virus of aclade other than clade
 1. 49. A method for the treatment or prophylaxisof influenza virus infections caused by H5N1 virus of a clade other thanclade 1, wherein the method comprises administration of atherapeutically effective amount of the combination of the H5 proteinwherein said H5 protein comprises a polypeptide sequence having at least98% sequence identity with the polypeptide sequence of SEQ ID NO:1 andan inactivated Newcastle disease virus combination to a subject in needof such a treatment.
 50. The method of claim 41, wherein saidadministration is a single-shot administration or a one doseadministration.
 51. A method of reducing the incidence of or severity ofinfluenza infection comprising the step of administering a compositionselected from the group consisting of: a. an H5 protein of clade 1 H5N1virus, wherein said H5 protein comprises or consists of a polypeptidesequence having at least 98% sequence identity with the polypeptidesequence of SEQ ID NO: 1, and b. a combination of the H5 protein ofgroup a) and an inactivated Newcastle disease virus.
 52. The H5 proteinof claim 1, for use in a method for reducing viral shedding in asubject, wherein said H5 protein is to be administered to a subjectinfected with or at risk of a viral infection with H5N1 virus of a cladeother than clade
 1. 53. The method of claim 52, further comprising useof an inactivated Newcastle disease virus in combination with the H5protein.
 54. Use of a H5 protein wherein said H5 protein comprises apolypeptide sequence having at least 98% sequence identity with thepolypeptide sequence of SEQ ID NO:1, for the preparation of a medicamentfor reducing viral shedding in a subject infected with or at risk of aviral infection with H5N1 virus of a clade other than clade
 1. 55. Themedicament of claim 54, further comprising use and an inactivatedNewcastle disease virus in combination with the H5 protein, wherein thecombination reduces viral shedding in a subject infected with or at riskof a viral infection with H5N1 virus of a clade other than clade 1.